Regenerative Therapy 10 (2019) 17e26

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Regenerative Therapy
journal homepage: http://www.elsevier.com/locate/reth

Original Article

Multilineage-differentiating stress-enduring (Muse)-like cells exist insynovial tissue
Eriko Toyoda a, Masato Sato a, *, Takumi Takahashi a, Miki Maehara a,
Yoshihiko Nakamura b, Genya Mitani a, Tomonori Takagaki a, Kosuke Hamahashi a,
Masahiko Watanabe aabDepartment of Orthopaedic Surgery, Surgical Science, Tokai University School of Medicine, 143 Shimokasuya, Isehara, Kanagawa 259-1193, Japan
Cell Processing Center, Tokai University Hospital, 143 Shimokasuya, Isehara, Kanagawa 259-1193, Japana r t i c l e i n f o

a b s t r a c t

Article history:
Received 30 May 2018
Received in revised form
30 September 2018
Accepted 23 October 2018

Introduction: Cartilage regeneration is a promising therapy for restoring joint function in patients withcartilage defects. The limited availability of autologous chondrocytes or chondrogenic progenitor cells isan obstacle to its clinical application. We investigated the existence and chondrogenic potential of synovial membrane-derived multilineage-differentiating stress-enduring (Muse)-like cells as an alternativecell source for cartilage regeneration.
Methods: Cells positive for stage-speciﬁc embryonic antigen-3 (SSEA-3), a marker of Muse cells, wereisolated from the synovial membranes of 6 of 8 patients (median age, 53.5 years; range 36e72 years) by
ﬂuorescence-activated cell sorting. SSEA-3-positive cells were cultured in methylcellulose to examine
their ability to form Muse clusters that are similar to the embryoid bodies formed by human embryonicstem cells. Muse clusters were expanded and chondrogenic potential of M-cluster-derived MSCs
examined using a pellet culture system. Chondrogenic differentiation was evaluated by proteoglycan,
safranin O, toluidine blue and type II collagen staining. To evaluate the practicality of the procedure forisolating Muse-like cells, we compared chondrogenic potential of M-cluster derived MSCs with expanded
cells derived from the clusters formed by unsorted synovial cells.
Results: Synovial membranes contained SSEA-3-positive cells that after isolation exhibited Muse-likecharacteristics such as forming clusters that expressed NANOG, OCT3/4, and SOX2. In the pellet culture system, cell pellets created from the M-cluster-derived MSCs exhibited an increase in wet weight,
which implied an increase in extracellular matrix production, displayed metachromasia with toluidine
blue and safranin O staining and were aggrecan-positive and type II collagen-positive by immunostaining. Unsorted synovial cells also formed clusters in methylcellulose culture, and the expanded cell
population derived from them exhibited chondrogenic potential. The histological and immunohistochemical appearance of chondrogenic pellet created from unsorted synovial cell-derived cells werecomparable with that from M-cluster-derived MSCs.
Conclusions: Muse-like cells can be isolated from the human synovial membrane, even from older patients, and therefore may provide a source of multipotent cells for regenerative medicine. In addition, thecluster-forming cell population within synovial cells also has excellent chondrogenic potential. Thesecells may provide a more practical option for cartilage regeneration.
© 2018, The Japanese Society for Regenerative Medicine. Production and hosting by Elsevier B.V. This is
an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Keywords:
Cartilage
Regenerative medicine
Chondrogenic potential
Multilineage-differentiating stress-enduring
cellsStage-speciﬁc embryonic antigens-3

Abbreviations: APC, allophycocyanin; BSA, bovine serum albumin; DAB, 3,30 -diaminobenzidine; DAPI, 40 ,6-diamidino-2-phenylindole; FBS, fetal bovine serum; FITC,
ﬂuorescein isothiocyanate; HRP, horseradish peroxidase; Ig, immunoglobulin; M-cluster, cluster cultured from synovialeMuse cells; MC, methylcellulose; aMEM, alphaminimum essential medium; MSC, mesenchymal stem cell; Muse, multilineage-differentiating stress-enduring cells; PBS, phosphate-buffered saline; PE, phycoerythrin;
SSEA-3, stage-speciﬁc embryonic antigen-3; SY-cluster, cluster cultured from unsorted synovial cells.
* Corresponding author.
E-mail addresses: etoyoda@tokai-u.jp (E. Toyoda), sato-m@is.icc.u-tokai.ac.jp (M. Sato), ttakahashi@tokai-u.jp (T. Takahashi), m-maehara@tsc.u-tokai.ac.jp (M. Maehara),kahiko@is.icc.u-tokai.ac.jp (Y. Nakamura), genya@syd.odn.ne.jp (G. Mitani), t-tom8@pc4.so-net.ne.jp (T. Takagaki), hamako@is.icc.u-tokai.ac.jp (K. Hamahashi), masahiko@is.
icc.u-tokai.ac.jp (M. Watanabe).
Peer review under responsibility of the Japanese Society for Regenerative Medicine.
https://doi.org/10.1016/j.reth.2018.10.005
2352-3204/© 2018, The Japanese Society for Regenerative Medicine. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license(http://creativecommons.org/licenses/by-nc-nd/4.0/).

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E. Toyoda et al. / Regenerative Therapy 10 (2019) 17e26

1. Introduction

2.2. Immunohistochemistry for SSEA-3

Hyaline cartilage regeneration is the ultimate goal in the care ofpatients with a cartilage defect including osteoarthritis. Hyaline
cartilage regeneration induced by chondrocyte sheet transplantation has been conﬁrmed in animal models of cartilage defects[1e4]. Based on these results, we have conducted a clinical study
using autologous knee chondrocyte sheets in patients with cartilage defects in the knee joint, including osteoarthritis patients.
Examination at the 1-year follow-up demonstrated hyaline cartilage regeneration at the transplanted site, and the outcomes of thisclinical study will be reported elsewhere.
Generally, autologous chondrocytes obtained from patients areused in cartilage regenerative medicine. In such procedures,
healthy cartilage tissue is sacriﬁced from healthy nonweightbearing sites, which can be scarce in the patient's joint. This restraint makes a second treatment by regenerative therapy impossible. In addition, adult human chondrocytes have low proliferative
capability, and they easily dedifferentiate and lose their chondrogenic characteristics during cultivation ex vivo. The limited availability of good-quality chondrocytes in a sufﬁcient number is one of
the major obstacles to the clinical implementation of cartilage
regenerative medicine.
The ideal solution to this chondrogenic cell shortage is to
identify easily accessible autologous cells that have both highproliferative capacity and chondrogenic potential. The existence of
nontumorigenic endogenous pluripotent-like stem cells, or
multilineage-differentiating stress-enduring (Muse) cells, in bonemarrow, skin, and adipose tissue has been reported recently [5e8].
Muse cells were originally identiﬁed as cells that are resistant tolong-term trypsin incubation and are known as cells that are
double positive for the pluripotent surface marker, stage-speciﬁcembryonic antigen-3 (SSEA-3), and CD105, and that have the capacity for self-renewal and triploblastic differentiation from a single cell [6]. The applicability of Muse cells for regenerative
treatments has been suggested in disease models of liver damage,stroke, skin ulcers related to diabetes mellitus, and osteochondral
defects [9e12].
This is the ﬁrst study to identify SSEA-3 and CD105 double
positive cells isolated from patient-derived synovial membrane,
which we designate as Muse-like cells and characterize thechondrogenic differentiation potential of their expanded cell, Mcluster-derived MSCs. Because patients who experience joint
problems sometimes undergo arthroscopic examination, whichallows for the collection of synovial membranes, we reasoned
that membranes collected from these patients may be a candidate autologous cell source for cartilage regenerative medicine.
We investigated whether M-cluster-derived MSCs have chondrogenic potential and their practicality for use in regenerative
medicine.

Synovial membranes were ﬁxed with 10% formalin solution
and parafﬁn embedded, and 10-mm thick sections were prepared.
For staining, the sections were deparafﬁnized and rehydrated, andincubated with 0.3% H2O2 in methanol for 30 min at room temperature to block endogenous peroxidase activity. The samples
were washed three times with phosphate-buffered saline (PBS)
for 10 min and incubated with blocking buffer (20% Block Ace; DS
Pharma Biomedical, Osaka, Japan), 5% bovine serum albumin(BSA; SigmaeAldrich, St Louis, MO, USA), and 0.3% Triton X-100 inPBS) for 30 min at room temperature. The slides were thenincubated with anti-SSEA-3 antibody (1:100, rat-anti-SSEA-3
antibody; MAB4303, Merck Millipore, Tokyo, Japan) diluted with
antibody dilution buffer overnight at 4  C. After the overnightincubation, the samples were washed three times with PBS for
10 min each at room temperature and incubated with secondary
antibody (1:200, horseradish peroxidase (HRP)-conjugated goatanti-rat immunoglobulin M (IgM); Jackson ImmunoResearch,West Grove, PA, USA) in dilution buffer for 2 h at room temperature. The samples were washed three times as before, incubatedwith 3,30 -diaminobenzidine (DAB; Wako Pure Chemical Industries, Osaka, Japan) solution for 20 min at room temperature,incubated with 0.01% H2O2 in DAB solution for 30 min at room
temperature, and ﬁnally washed with distilled water. Counterstaining was performed with Mayer's hematoxylin dehydrate(Wako Pure Chemical Industries).

2. Methods
2.1. Ethics statement and synovial membrane preparation
Experiments using human synovial membranes were performed with the approval and guidance of the Clinical Research
Review Committee of Tokai University School of Medicine(approval number: 09R-070, 10I-52, 13I-33). Debrided synovial
tissues were obtained from the knee joints of patients during
surgery for total knee arthroplasty, high tibial osteotomy, or
treatment of meniscus tear. The patients’ median age was 53.5
years, their age range was 36e72 years, and ﬁve were male andthree were female. Written informed consent was obtained fromall patients.

2.3. Isolation of synovial cells
Synovial membranes were rinsed with saline, ﬁnely minced, anddigested with 5 mg/mL collagenase type I (Worthington Biochemical Corp., Lakewood, NJ, USA) in growth medium (alpha-minimum
essential medium (aMEM; SigmaeAldrich, Tokyo, Japan) containing
10% fetal bovine serum (FBS; AusGeneX, Oxenford, Australia) and1  kanamycin (Thermo Fisher Scientiﬁc, Kanagawa, Japan) at 37  C.
Dispersed synovial cells were collected by centrifugation, washedtwice with PBS (Thermo Fisher Scientiﬁc), and seeded at
0.5e2.0  104 cells/cm2 in culture ﬂasks with growth medium.
Nonadherent cells were removed by medium replacement.
Adherent cells were cultured until they reached subconﬂuence andwere then dissociated using TripLE Express (Thermo Fisher Scientiﬁc). Aliquots of the obtained cells were suspended in Cellbanker 1(Zenoaq, Fukushima, Japan) and cryopreserved for later use.
2.4. Fluorescence-activated cell sorting and surface marker
expression analysis
Cryopreserved synovial cells were thawed, seeded in growth
medium in culture ﬂasks, and expanded until they reached
conﬂuence. Synovial cells were passaged once or twice, dependingon the number of cells obtained, and then sorted to isolate SSEA-3positive cells. To isolate cells that were double positive for SSEA-3
and CD105, synovial cells were dissociated from culture dishes using TripLE Express and suspended in FACS Buffer (PBS containing
0.2% BSA and 1 mM ethylenediaminetetraacetic acid), incubatedwith mouse phycoerythrin-conjugated anti-CD105 antibody (1:20;
Beckman Coulter, Tokyo, Japan) and rat anti-SSEA-3 IgM antibody(1:50; Merc Millipore), and then incubated with ﬂuorescein isothiocyanate (FITC)-conjugated anti-rat IgM (Jackson ImmunoResearch) as described previously [6,13]. Synovial cells wereincubated with FITC-conjugated anti-rat IgM and PE-conjugated IgG(Beckman coulter), and they were used to set sorting ﬂuorescentthresholds. Cells double positive for CD105 and SSEA-3 were sorted
using a FACSVantage (BD Biosciences, Tokyo, Japan). A portion of theE. Toyoda et al. / Regenerative Therapy 10 (2019) 17e26

synovial cells was reserved before immunostaining as unsorted
synovial cells to compare the efﬁciency of isolation with that ofMuse cells for use in the preparation of chondrogenic cells.
2.5. Cluster formation in methylcellulose medium
Cluster formation by SSEA-3 and CD105 double-positive cells
and unsorted synovial cells was induced by culturing the cells inmethylcellulose (MC) using MethoCult H4230 (MC medium supplemented with FBS, BSA, and 2-mercaptoethanol; StemCell Technologies, Vancouver, Canada) using a slight modiﬁcation of amethod described previously [13]. Muse cells or unsorted synovial
cells were suspended at a concentration of 1000e10000 cells/mL in
MethoCult H4230 and seeded in 35-mm culture dishes. After 7e10
days of MC culture, the number of clusters with a diameter larger
than 25 mm formed from synovial Muse cells (M-clusters) or unsorted synovial cells (SY-clusters) were counted under a microscope. The cluster-forming rate was calculated as the number ofclusters per number of cells plated in each dish and was shown as
the mean ± standard deviation calculated from three technicalreplicates.
2.6. Immunocytochemistry of clusters
The expression of pluripotent markers by M and SY-clusters
was assessed using a slight modiﬁcation of a previously describedmethod [6]. Clusters were collected from MC cultures, resuspended
in aMEM, and washed three times with PBS. Clusters were suspended in PBS at a concentration of 500e1000 clusters/mL, and
0.1 mL of the cluster suspension (50e100 clusters/slide) was
centrifuged onto the surface of a glass slide using a Cytospin™ 4
cytocentrifuge (Thermo Fisher Scientiﬁc) at 500 rpm for 5 min. The
slides were dried with cold air and ﬁxed with 4% (w/vol) paraformaldehyde in 0.1 M phosphate buffer. Before immunostaining,clusters were permeabilized with 0.5% Triton X-100 in 0.02 M PBS
for 5 min at room temperature and incubated with blocking solution (5% normal goat serum in 0.02 M PBS) for 30 min at room
temperature. The blocking solution was discarded, and the slides
were incubated with the following primary antibodies: polyclonal
rabbit anti-NANOG antibody (1:500; Merck Millipore), polyclonal
rabbit anti-OCT3/4 antibody (1:100; Santa Cruz Biotechnology,
Dallas, TX, USA), and polyclonal rabbit anti-SOX2 antibody (1:500;
Abcam, Cambridge, UK) in antibody diluent (1% normal goat serum
and 0.1% Triton X-100 in 0.02 M PBS) at 4  C overnight. Rabbit IgG(1:250; Agilent Technologies, Santa Clara, CA, USA) was used as theisotype control. Alexa Fluor488-conjugated goat anti-rabbit IgG
secondary antibody (1:1000; Thermo Fisher Scientiﬁc) was used for
detection. Nuclei were detected using ProLong® Diamond Antifade
Mountant with 40 ,6-diamidino-2-phenylindole (DAPI; Thermo
Fisher Scientiﬁc). Images were acquired with a BZ-X7000 ﬂuorescence microscope (Keyence, Osaka, Japan).
2.7. In vitro chondrogenesis
Chondrogenic differentiation of cells in pellet culture wasassessed as described previously with slight modiﬁcations [14,15].
M and SY-clusters were collected from MC medium, suspended in
aMEM medium, washed, and seeded in growth medium in culture
ﬂasks to obtain a sufﬁcient number of cells for culture. Cells wereexpanded for 1e2 weeks, depending on the number of clusters
produced, and harvested using TripLE Express. Two-hundred ﬁfty
thousand cells obtained from the expanded M or SY-clusters wereplaced in a 15 mL polypropylene tube (BD Biosciences) in 400 mL ofchondrogenic medium (high-glucose Dulbecco's modiﬁed Eagle
medium; Gibco, Thermo Fisher Scientiﬁc) supplemented with

19

100 mg/mL sodium pyruvate (Wako Pure Chemical Industries),
100 nM dexamethasone (SigmaeAldrich), 40 mg/mL L-proline(SigmaeAldrich), 50 mg/mL ascorbate-2-phosphate (Nissin Pharmaceutical Co., Ltd., Yamagata, Japan), 1/100 (vol/vol) ITS þ Premix(Corning, Corning, NY, USA), 100 ng/mL recombinant human BMP2/
BMP6 heterodimer (R&D Systems, Minneapolis, MN, USA), and10 ng/mL transforming growth factor-b1 (PeproTech, Rocky Hill, NJ,
USA). The cells were centrifuged at 600g for 10 min. The tubes
were left standing in an incubator at 37  C with 5% CO2 for 20e21
days or 28 days, during which the medium was changed every 3e4days. The cell pellets were weighed every week and harvested at 21
days or 28 days for histological examination.
The cell pellets were ﬁxed in 4% paraformaldehyde in PBS for
20 min and embedded in Tissue-Tek O.C.T. Compound (Sakura
Finetech, Tokyo, Japan) and frozen at 80  C. Frozen sections were
cut at 10-mm thickness at 15  C on a cryostat and mounted onto
glass slides, air dried, and ﬁxed with 4% paraformaldehyde in
0.01 M phosphate buffer for 30 min at room temperature. For
histological examination, sections were stained with hematoxylin
and eosin, safranin O, and toluidine blue according to standard
protocols. For immunohistochemical analysis, sections were incubated with blocking solution (0.3% Triton X-100 in BlockAid
Blocking Solution (Thermo Fisher Scientiﬁc)) for 45 min at room
temperature, after which the blocking solution was discarded andthe slides were incubated with the following primary antibodies:goat anti-human aggrecan (1:10; Human Mesenchymal Stem Cell
Functional Identiﬁcation Kit, R&D Systems), and goat anti-humancollagen 1 (1:200; SouthernBiotech, Birmingham, AL, USA) inblocking solution at 4  C overnight. Alexa Fluor546-conjugateddonkey anti-goat antibody (1:400; Thermo Fisher Scientiﬁc) wasused as secondary antibody for detection. Nuclei were detectedusing ProLong® Diamond Antifade Mountant with DAPI (Thermo
Fisher Scientiﬁc). For type II collagen detection, sections wereincubated with 0.4% pepsin (DAKO, Glostrup, Denmark) at 37  C for
30 min and washed in distilled water, followed by incubation in
0.3% hydrogen peroxide/methanol solution at RT for 15 min. Afterwashing with PBS, sections were incubated with a diluted primaryanti-human type II collagen antibody (1:100; F-57: Daiichi Fine
Chemical, Toyama, Japan) overnight at 4  C, followed by incubation
with the ImmPRESS Reagent Anti Mouse Ig (Vector Laboratories,
Burlingame CA) at room temperature. Finally, the sections were
stained with DAKO Liquid DAB substrate chromogen system(DAKO) and counterstained with hematoxylin. Images were acquired with a BZ-X7000 ﬂuorescence microscope (Keyence).

2.8. Surface marker expression
SY-cluster-derived cells were analyzed using ﬂow cytometry at
the same time as they were used for in vitro chondrogenesis.
Expanded SY-cluster-derived cells were harvested using TripLE
Express, suspended in FACS Buffer, and immunostained with thefollowing antibodies: CD31eFITC (clone: 5.6E), CD45eFITC (clone:J.33), and CD105ePE (clone:1G2) from Beckman Coulter;
CD81eallophycocyanin (APC) (clone: JS-81), CD90eAPC (clone:
5E10), CD49aePE (clone: SR84), CD106eFITC (clone: 51-10C9),
CD44eFITC (Clone: G44-26), CD34ePE (clone: 563), and CD271ePE(clone: C40-1457) from BD Biosciences; CD146ePE (clone: F4-35H7(S-Endo 1)) from BioCytex (Marseille, France); and STRO-1eFITC
from BioLegend (San Diego, CA, USA). SSEA-3 was identiﬁed bystaining as described above. Fluorochrome-labeled anti-mouse
IgG1 antibody (clone: 679.1Mc7, Beckman Coulter) was used as anegative control. Stained cells were analyzed using a FACSVerse (BD
Biosciences), and data were analyzed using FlowJo software (TomyDigital Biology, Tokyo, Japan).

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E. Toyoda et al. / Regenerative Therapy 10 (2019) 17e26

3. Results
3.1. Existence of SSEA-3-positive cells in the human synovial
membrane
To identify the presence of Muse cells, sections of human synovial membrane were examined by immunostaining using antiSSEA-3 antibody (Fig. 1). Similar to previous reports for adipose
tissue [7] and the dermis [6], synovial membranes obtained fromthe knee joint of patients contained SSEA-3-positive cells. SSEA-3positive cells were found in both the connective tissue and adipose
tissue areas of the synovial membrane. They were scattered in theconnective tissue and did not appear to be related to particular
histological structures such as vessels.
3.2. Identiﬁcation of muse-like cells among synovial cells
Muse cells, which exhibit self-renewal capacity and the ability todifferentiate triploblastic lineage-cells from a single cell, are known
to be enriched by cell sorting using anti-SSEA-3 antibody. To isolate
SSEA-3- and CD105-double-positive (Muse-like) cells by
ﬂuorescence-activated cell sorting, synovial membrane tissues
were dispersed by enzymatic digestion and the adherent cells wereused.
Adherent synovial membrane-derived cells exhibited spindleshaped ﬁbroblast-like features (Fig. 2a). These synovial cells were
cultivated until conﬂuent and were then used to Muse-like cell
preparation. Cell sorting for SSEA-3-positive cells was performed as
described previously [6]. Almost all synovial cells that wereexpanded ex vivo expressed CD105 antigen (Fig. 2b). Table 1 provides a summary of the Muse-like cells isolated from the samples

from 8 patients aged from 36 to 72 years (ﬁve males and threefemales). The percentage of SSEA-3-positive cells among the synovial membrane-derived cells varied between patients andranged from 0.05% to 8.78% (mean ± SD: 2.4% ± 2.8%). We found no
relationship between the percentage of SSEA-3-positive cells andpatient age. Passage 1 cells tended to contain a higher percentage of
SSEA-3-positive cells than did passage 2 cells, which suggests that alonger culture before sorting decreases the percentage of SSEA-3positive cells. Therefore, we did not expand the synovial cells
past passage 2 to scale up Muse-like cell preparation. Samples fromtwo patients contained a particularly low percentage of SSEA-3positive cells; therefore, cell sorting and cluster formation was
not performed.
Muse cells have been shown to form clusters similar to theembryoid bodies formed by human embryonic stem cells when
cultured in suspension culture [5e8]. To evaluate the characteristics of the SSEA-3-positive cells isolated from synovial cells,
they were cultured in MC according to the protocol for M-cluster
formation described by Kuroda et al. [13] and the clusters wereanalyzed. After 7e10 days of MC culture, Muse-like cells obtained from all patients formed embryoid body-like clusters (Mclusters) (Fig. 2d). Among the Muse-like cells from six patients,
4%e20% of SSEA-3-positive cells formed M-clusters in MC culture (average: 10.6% ± 6.1%). The estimated content of M-cluster
forming cells for unsorted synovial cells ranged from 0.1 to 1.1%,
as estimated by the multiplication of the percentage of SSEA-3
positive cells and the corresponding percentage of M-cluster
forming cells (Table 1).
To compare the efﬁciency of isolation of Muse-like cells, unsorted synovial cells, which were thought to contain Muse-likecells, were also subjected to MC culture.

Fig. 1. SSEA-3-positive cells in the synovial membrane. A Representative immunostaining of synovium sections stained with anti-SSEA-3 antibody. B Serial section of A stained withhematoxylin and eosin. SSEA-3-positive cells were found in both adipose tissue and connective tissue. C Magniﬁed image of the area enclosed in the box in A. Arrowheads indicate
SSEA-3-positive cells. D Counterstaining of image C with Mayer's hematoxylin.

E. Toyoda et al. / Regenerative Therapy 10 (2019) 17e26

21

Among the unsorted synovial cells, 3%e20% of cells formedclusters in MC culture (SY-cluster) (Fig. 2c, Table 1). The estimatednumber of M-cluster forming cells in unsorted synovial cells was
lower than that of SY-clusters, suggesting that the SSEA-3-positive
cells were once damaged by laser irradiation during FACS isolationand their cluster forming rate was decreased compared to unsorted synovial cells. Alternatively, SY-clusters might consist of
not only Muse-like cell-derived clusters but also clusters formed
from SSEA-3-negative cells. We next evaluated the characteristicsof these clusters further.
3.3. Evaluation of the pluripotent marker expression of M and SYcluster-forming cells
Clusters formed from bone marrow, dermis, and adipose tissue
derived Muse cells can express NANOG, SOX2, and OCT3/4 andexhibit triploblastic differentiation [5e8]. Immunostaining wasused to examine the expression of pluripotent markers in M- andSY-clusters. M- and SY-clusters were collected from MC cultures
and attached to glass slides. Immunohistochemical staining for
NANOG, SOX2, and OCT3/4 revealed that both synovial membranederived M- and SY-clusters expressed these markers (Fig. 3).
Because of the overlapped cells on the slides, the outlines of speciﬁcgreen signals for markers were unclear; however, colocalization ofbrighter green signals and DAPI signals indicated that the markers
are located in the nucleus.
3.4. Comparison of chondrogenic potency of M and SY-clusterderived cells

Fig. 2. Procedures to prepare M-cluster-derived MSCs and SY-cluster-derived cells.
Clusters formed from the unsorted cell population were named SY-clusters and thosefrom SSEA-3 and CD105 double-positive populations were named M-clusters. Photographs show typical phaseecontrast microscopic appearance. a Synovial cells cultured
in a plastic dish. b Representative dot plot of SSEA-3 and CD105 double-positive cellsorting. c, d A cluster formed in MC medium from unsorted synovial cells (c) anddouble-positive cells (d). eeh Expansion cultures on day 1 (e, f) and day 15 (g, h) of SYcluster-derived cells (e, g) or M-cluster-derived MSCs (f, h). Clusters were collectedfrom MC and seeded on plastic dishes. Both cluster-forming cells showed good proliferative capacity.

One of our aims was to assess Muse-like cells as a potential cellsource for cartilage regeneration, which means that a sufﬁcientnumber of cells must be obtained from the cell preparation procedures. Because of the shortage of M-cluster-forming cells, theyneeded to be expanded to obtain a sufﬁcient number of cells for
performing in vitro chondrogenic differentiation. The higher ratioof SY-cluster forming cells in synovial cells and they can be prepared without damaging by FACS sorting suggested that unsorted
synovial cells may also be a potential cell source. To compare thechondrogenic potency of cells derived from M- and SY-clusters,
both type of clusters were expanded for the same culture period.
Both the M- and SY-cluster-forming cells proliferated actively andthis generated M-cluster-derived MSCs and SY-cluster-derived
cells, respectively (Fig. 2e -h).
In vitro chondrogenesis was examined using M-cluster-derived
MSCs and SY-cluster-derived cells from same patient (Fig. 4A). Both

Table 1
Summary of Muse-like cell isolation from synovium and cluster formation in MC culture.
Patient

Age

GenderPassage

SSEA-3þ CD105þ cells
(%)

1
2
3
4
5
6
7
8

56
42
59
50
71
36
51
72

F
M
F
M
M
M
M
F

P1
P1
P1
P1
P2
P2
P2
P2

a

8.78
2.1
1.86
3.67
1.2
1
0.76
0.05

M-cluster forming
rate (%)b

Estimation of M-cluster forming
rate in unsorted synovial cells (%)

12.3 ± 0.35
14 ± 0.6
20.1 ± 1.1
4.34 ± 0.11
4.74 ± 0.80
8.02 ± 2.05
NT
NT

1.08
0.29
0.37
0.16
0.06
0.08
NT
NT

NT: Not tested.
a
The percentage of SSEA-3þ CD105þ cells based on FACS sorting.
b
The percentage of cluster forming cells in SSEA-3þ CD105þ.sorted cells obtained from cluster formation in MC culture.
c
Cluster forming rate in unsorted synovial cells obtained from cluster formation in MC culture.

SY-cluster forming cells
in synovial cells (%)c

NT
NT
NT
NT
6.80 ± 0.37
3.31 ± 0.55
20.2 ± 1.30
19.6 ± 4.19

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E. Toyoda et al. / Regenerative Therapy 10 (2019) 17e26

Fig. 3. Expression of stemness markers by M- and SY-clusters. Immunostaining of clusters formed from Muse-like cells and unsorted synovial cells. Clusters were positive for
NANOG, OCT3/4, and SOX2. Scale bars ¼ 50 mm. DAPI was used as a counterstain.

cell pellets created from M-cluster-derived MSCs and SY-clusterderived cells exhibited an increase in wet weight with culture
time (Fig. 4B), which implied an increase in extracellular matrix
production [16]. Cell pellets created from the SY-cluster-derived cellfrom other patients also exhibited an increase in wet weight with
culture time (Fig. 4C). Chondrogenic differentiation of pellets was
conﬁrmed by safranin O staining, toluidine blue staining, and immunostaining of aggrecan at 21 days (Fig. 4D and E) and type II
collagen at day 28 days (Fig. 4F) after chondrogenic induction. BothM-cluster-derived MSCs and SY-cluster-derived cells generated
pellets that displayed metachromasia with toluidine blue andsafranin O staining and were aggrecan-positive and type II collagen-positive by immunostaining. Type I collagen staining was also
detected, especially in the surface region of pellets (Fig. 4E).
The increase in the weight of pellets and their histological andimmunohistochemical appearance did not seem to differ between
M-cluster-derived MSCs and SY-cluster-derived cells.

3.5. Phenotypic characterization of SY-cluster-derived cells
As a cell source for cartilage regeneration, the SY-cluster-derived
cell population that is obtained from cluster formation of synovial
cells in semisolid culture without SSEA-3-sorting may be a practical
option. To characterize the expanded SY-cluster-derived cells, theexpression of surface markers was analyzed using ﬂow cytometryat the same time as their chondrogenic potential was evaluated. We
could not perform this analysis using M-cluster-derived cells
because of their limited availability. Expanded SY-cluster-derived
cells did not express the hematopoietic marker CD45 or theendothelial marker CD31, but they did express mesenchymal stem
cell (MSC) markers CD90, CD105, and CD44. A small subset of cells
expressed the hematopoietic stem cell marker CD34, the lowafﬁnity nerve growth factor receptor CD271, CD146 (M-CAM),
CD106 (V-CAM-1), another MSC marker STRO-1, and SSEA-3(Fig. 5).

E. Toyoda et al. / Regenerative Therapy 10 (2019) 17e26

23

Fig. 4. Chondrogenic differentiation of M-cluster-derived MSCs and SY-cluster-derived cells. A Representative gross appearance of pellets cultured with a 1-mm scaled ruler. B
Increase in the wet weight of pellets created from M-cluster-derived MSCs and SY-cluster-derived cells obtained from one patient. C Wet weight of pellets created from SY-clusters-derived cells from 3 individual patients. Values are the mean and SD of three pellets created from each source from each patient. D Representative histological staining withhematoxylin and eosin (HE), safranin O (Saf-O), and toluidine blue (TB). E-F Immunohistochemical examination of chondrogenic differentiation of pellets at d21(e) or d28(f). Scalebars in d, e and f ¼ 300 mm.

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E. Toyoda et al. / Regenerative Therapy 10 (2019) 17e26

Fig. 5. Phenotypic characterization of SY-cluster-derived cells. Expression of cell surface markers was analyzed by ﬂow cytometry. Values are the mean and SD of thepercentage expression of each cell surface marker obtained from SY-cluster-derived
cells from four patients.

4. Discussion
Muse cells have been described as stress-enduring clusterforming cells with multipotency. In this report, we investigated theexistence of Muse-like cells in the synovial membrane. Kuroda et al.
reported that the percentage of Muse cells was 1.3% ± 0.1% in naive
human ﬁbroblasts and 1.1% ± 0.1% in human mesenchymal stromalcells [6]. They also reported that the percentage of Muse cells in theadherent fraction of bone marrow aspirate was 0.3% ± 0.08% for ﬁrst
passage, 0.5% ± 0.04% for second passage, and 0.9% ± 0.1% for ﬁfth
passage cells. They also conﬁrmed the SSEA-3 and CD105 doublepositive phenotype as a marker of Muse cells and reported that
more than half of the double-positive cells obtained from humanbone marrow-derived MSCs and human ﬁbroblasts formed Mclusters in suspension culture. Ogura et al. found a frequency of
3.8% ± 0.9% SSEA-3-positive cells in purchased adipose MSCs and8.8% ± 1.3% in freshly isolated subcutaneous adipose MSCs. They
also reported that 30e40% of SSEA-3-positive adipose-Muse cells
formed M-clusters in a single suspension culture but that no cluster
formation was observed in SSEA-3-negative adipose tissue-derived
MSCs [7]. Heneidi et al. also reported Muse cells in adipose tissue
and that an adipose tissue-derived Muse cell population could be
prepared under highly stressed conditions [5]. This population
contained about 90% SSEA-3- and CD105-positive cells.
In this report, we used the SSEA-3- and CD105-double-positive
phenotype to identify Muse-like cells obtained from synovial
membrane-derived cells. We conﬁrmed that the synovial-derived
adherent cells passaged once or twice also contained SSEA-3 andCD105 double-positive cells at an average frequency of 2.4% ± 2.8%
(range 0.05e8.78%). Although we did not examine the multilineage
differentiation and self-renewal capacity of the Muse-like cells,
0.3% ± 0.4% of these cells obtained from the synovial cell population
exhibited Muse-like characteristics as shown by their SSEA-3 positivity and their formation of clusters in MC culture that express
NANOG, SOX2 and OCT3/4. Therefore, the frequency of Muse-likecells in the synovial membrane might be similar to that in othertissues, human ﬁbroblasts, and bone marrow but may be less thanthat of adipose tissue. We also found that the synovial membranes
obtained from older osteoarthritis patients contained Muse-likecells. In general, although the somatic stem cell number declines
with age [17e19], our results suggest that synovial Muse-like cells
may be a potential autologous cell source for cartilage regeneration
and regenerative therapy in older patients. Considering that the
low yield of Muse-like cells may be caused by cell damage
accompanying FACS sorting, an alternative procedure for preparation of SSEA-3-positive cells may improve the yield of Muse-likecells. The superior effect of intra-articular injection of bonemarrow-derived Muse cells compared to non-Muse cells was previously reported in an osteochondral defect model [12]. Instead ofMuse cells obtained from bone marrow aspirates, synovial membrane derived Muse-cells is another promising option for thetreatment of osteochondral defects, whereas unsorted SY-cluster
derived cells may not be suitable.
The cluster-forming rates of SSEA-3 and CD105 double-positive
cells derived from synovial membranes were low compared withthose of Muse cells derived from other tissues. Given the deﬁnitionof Muse cells as cells exhibiting stress-enduring characteristics, theuse of more stringent cell preparation processes before cell isolation using SSEA-3 may improve the yield of Muse-like cells [5,6].
We found that unsorted synovial cells also formed clusters
when cultured under the same conditions, and that they also
exhibited an OCT3/4-, NANOG-, and SOX2-positive phenotype on
immunostaining, which implies multipotency. This cell populationis thought to be a mixture of Muse-like cells and non-Muse cells.
We found a higher cluster-forming cell number would be obtained
from unsorted synovial cells than from sorted Muse-like cells. The
estimated number of SSEA-3-positive cells in the unsorted cell
cultures (0.05%~8.78%) was less than that of clusters formed fromunsorted synovial cells (3.31%~20.2%). There is a possibility that theisolation of SSEA-3-positive cell by FACS sorting damaged Muselike cells and their cluster forming rate decreased. Alternatively,
this suggests that SY-clusters were formed to some extent fromboth Muse-like cells and non-Muse cells. In addition, the clusterforming ratios of SSEA-3-positive cells and unsorted synovial cells
from the same individual were related. These results imply thatcells with the ability to form clusters in semisolid culture conditions exist among synovial cells and that this ability is independentof the SSEA-3-positive phenotype.
These cluster-forming cells from unsorted synovial cells also
exhibited similar chondrogenic potential to those from sorted cells.
We conﬁrmed their chondrogenic differentiation by an increase in
wet weight of pellets and an aggrecan-positive and type II collagenpositive phenotype. Because the expression of type II collagentends to lag behind that of type I collagen [20] and increases
continuously during chondrogenic maturation of the cell pellet inculture [16], we reasoned that the 28-day differentiation period
might have been needed for detecting type II collagen produced
under our immunostaining conditions.
The preparative procedures for clinical application should
ensure a balance between chondrogenic potency and cell yield. In
this study, we found that almost all expanded SY-cluster-derived
cells that can be obtained by simply culturing synovial cells insemisolid condition expressed CD90, CD105, CD44 and exhibitedchondrogenic potential. We have reported that adult knee chondrocyte sheets that demonstrated the efﬁcacy in hyaline cartilage
regeneration also expressed CD90 and CD44 [21]. Further, our results agree with previous ﬁndings that CD90 and CD105 aremarkers of cells with excellent chondrogenic potential within the
synovial tissue [22e25]. Positivity of CD90, CD44 and CD105 of SYcluster -derived cells support the idea that the cells can be potential
alternative cell source for cartilage regeneration. It is possible thatthe cell populations in the unsorted synovial cells that exhibitedchondrogenic potential could be the same population as the reported synovial-derived chondrogenic cells [26e29]. SY-cluster-E. Toyoda et al. / Regenerative Therapy 10 (2019) 17e26

derived cells contained a small population of cells that expressed
CD34, CD271, CD146, CD106, or STRO-1. Variation in the ratios ofthese subsets between patients may be an important factor determining the balance between chondrogenic potential and the risk of
unexpected lineage differentiation. SY-cluster-derived cells also
contained a few percent of SSEA-3-positive cells, which may be
self-renewing Muse-like cells.
We found that the unsorted synovial cells formed clusters that
expressed the proteins NANOG, OCT-3/4, and SOX2. Bone marrowderived MSCs from young rats express stemness markers (Oct 4,
Sox 2, and Nanog) and exhibit chondrogenic, osteogenic and adipogenic potential [23]. Wang et al. reported that human ectopic
expression of OCT-4 in MSCs from amniotic ﬂuid can upregulate
NANOG and SOX-2 expression and that this is associated with anincrease in colony-forming ability [30]. Although further study isneeded, we believe that these markers expression of SY-clusters
might represent the properties of synovial MSCs elicited undersemisolid culture conditions. Chondrocytes can proliferate insemisolid culture conditions and require three-dimensional conditions to maintain their chondrogenic properties [31,32]. Therefore, it seems that the process of cluster formation in semisolid
culture places selective pressure on cells with chondrogenic
potential.
Our results have indicated that arthroscopically collectable synovial membranes, even from older patients, provide one practicalcell source for obtaining autologous Muse-like cells in situations
where a small number of the cells is sufﬁcient and multipotency isrequired. When considered for application in tissue engineering forregenerative medicine, cells with high proliferative and monodifferentiating potential toward the expected lineage are ideal.
Multipotency is inseparably linked to the risk of unexpected celldifferentiation. Precise procedures and meticulous monitoring ofcell differentiation are needed if multipotent cells are to be used inhuman therapy. In this context, SSEA-3 selection is dispensable forchondrogenic cell preparation from synovial membrane-derived
cells. Instead, cluster-forming synovial cells may be applicable asa cell source for cartilage tissue engineering. This would ensureboth chondrogenic potency and availability.
5. Conclusions
Muse-like cells can be isolated from the human synovial
membrane, even from older patients, and thus may provide a
multipotent cell source for regenerative medicine. Additionally, theexpanded cells derived from cluster-forming cell within the synovial cells seems to contain Muse-like cells, also has excellent
chondrogenic potential and may provide a more practical option for
cartilage regeneration.
Conﬂicts of interest
The authors declare that they have no competing interests.
Funding
Not applicable.
Acknowledgements
We would like to express our gratitude to Yoshihiko Kushida
and Mari Dezawa, Department of Stem Cell Biology and Histology,
Tohoku University Graduate School of Medicine, for assistance withthe histological examination and comments that greatly improved
the manuscript. We are grateful to the Education and Research

25

Support Center, Tokai University, for providing expertise that
greatly assisted the research.
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