Scholarly article on topic 'Not single but periodic injections of synovial mesenchymal stem cells maintain viable cells in knees and inhibit osteoarthritis progression in rats'

Not single but periodic injections of synovial mesenchymal stem cells maintain viable cells in knees and inhibit osteoarthritis progression in rats Academic research paper on "Biological sciences"

CC BY-NC-ND
0
0
Share paper
Academic journal
Osteoarthritis and Cartilage
OECD Field of science
Keywords
{Osteoarthritis / "Mesenchymal stem cells" / Synovium / "Trophic factors" / "Periodic injections"}

Abstract of research paper on Biological sciences, author of scientific article — N. Ozeki, T. Muneta, H. Koga, Y. Nakagawa, M. Mizuno, et al.

Summary Objective We investigated the effects of single or repetitive intra-articular injections of synovial mesenchymal stem cells (MSCs) on a rat osteoarthritis (OA) model, and elucidated the behaviors and underlying mechanisms of the stem cells after the injection. Design One week after the transection of the anterior cruciate ligament (ACL) of wild type Lewis rats, one million synovial MSCs were injected into the knee joint every week. Cartilage degeneration was evaluated with safranin-o staining after the first injection. To analyze cell kinetics or MSC properties, luciferase, LacZ, and GFP expressing synovial MSCs were used. To confirm the role of MSCs, species-specific microarray and PCR analyses were performed using human synovial MSCs. Results Histological analysis for femoral and tibial cartilage showed that a single injection was ineffective but weekly injections had significant chondroprotective effects for 12 weeks. Histological and flow-cytometric analyses of LacZ and GFP expressing synovial MSCs revealed that injected MSCs migrated mainly into the synovium and most of them retained their undifferentiated MSC properties though the migrated cells rapidly decreased. In vivo imaging analysis revealed that MSCs maintained in knees while weekly injection. Species-specific microarray and PCR analyses showed that the human mRNAs on day 1 for 21 genes increased over 50-fold, and increased the expressions of PRG-4, BMP-2, and BMP-6 genes encoding chondroprotective proteins, and TSG-6 encoding an anti-inflammatory one. Conclusion Not single but periodic injections of synovial MSCs maintained viable cells without losing their MSC properties in knees and inhibited osteoarthritis (OA) progression by secretion of trophic factors.

Academic research paper on topic "Not single but periodic injections of synovial mesenchymal stem cells maintain viable cells in knees and inhibit osteoarthritis progression in rats"

Osteoarthritis and Cartilage xxx (2016) 1—10

Not single but periodic injections of synovial mesenchymal stem cells maintain viable cells in knees and inhibit osteoarthritis progression in rats

N. Ozeki ft, T. Muneta §, H. Koga §, Y. Nakagawa §, M. Mizuno f, K. Tsuji ||, Y. Mabuchi t, C. Akazawa t, E. Kobayashi #, K. Matsumoto ff, K. Futamura ff, T. Saito Z, I. Sekiya f *

f Center for Stem Cell and Regenerative Medicine, Tokyo Medical and Dental University, Tokyo, 113-8510, Japan t Department of Orthopaedic Surgery, Yokohama City University, Yokohama, 236-0004, Japan

§ Department of Joint Surgery and Sports Medicine, Graduate School, Tokyo Medical and Dental University, Tokyo, 113-8510, Japan || Department of Cartilage Regeneration, Graduate School, Tokyo Medical and Dental University, Tokyo, 113-8510, Japan t Department of Biochemistry and Biophysics, Graduate School, Tokyo Medical and Dental University, Tokyo, 113-8510, Japan # Department of Organ Fabrication, Keio University School of Medicine, Tokyo, 160-8582, Japan

ff Department of Allergy and Clinical Immunology, National Research Institute for Child Health and Development, Tokyo, 157-8535, Japan

ARTICLE INFO

SUMMARY

Article history: Received 24 April 2015 Accepted 27 December 2015

Keywords: Osteoarthritis Mesenchymal stem cells Synovium Trophic factors Periodic injections

Objective: We investigated the effects of single or repetitive intra-articular injections of synovial mesenchymal stem cells (MSCs) on a rat osteoarthritis (OA) model, and elucidated the behaviors and underlying mechanisms of the stem cells after the injection.

Design: One week after the transection of the anterior cruciate ligament (ACL) of wild type Lewis rats, one million synovial MSCs were injected into the knee joint every week. Cartilage degeneration was evaluated with safranin-o staining after the first injection. To analyze cell kinetics or MSC properties, luciferase, LacZ, and GFP expressing synovial MSCs were used. To confirm the role of MSCs, species-specific microarray and PCR analyses were performed using human synovial MSCs. Results: Histological analysis for femoral and tibial cartilage showed that a single injection was ineffective but weekly injections had significant chondroprotective effects for 12 weeks. Histological and flow-cytometric analyses of LacZ and GFP expressing synovial MSCs revealed that injected MSCs migrated mainly into the synovium and most of them retained their undifferentiated MSC properties though the migrated cells rapidly decreased. In vivo imaging analysis revealed that MSCs maintained in knees while weekly injection. Species-specific microarray and PCR analyses showed that the human mRNAs on day 1 for 21 genes increased over 50-fold, and increased the expressions of PRG-4, BMP-2, and BMP-6 genes encoding chondroprotective proteins, and TSG-6 encoding an anti-inflammatory one. Conclusion: Not single but periodic injections of synovial MSCs maintained viable cells without losing their MSC properties in knees and inhibited osteoarthritis (OA) progression by secretion of trophic factors.

© 2016 The Authors. Published by Elsevier Ltd and Osteoarthritis Research Society International. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/

4.0/).

Introduction

Osteoarthritis (OA) is the most prevalent degenerative joint disease, associated with multiple risk factors such as age, sex, mal-

* Address correspondence and reprint requests to: I. Sekiya, Center for Stem Cell and Regenerative Medicine, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8510, Japan. Tel.: 81-3-5803-4017; fax: 81-3-5803-0192. E-mail address: sekiya.arm@tmd.ac.jp (I. Sekiya).

alignment, obesity, genetic factors, and trauma1. Although the incidence of OA is rising due to aging populations2, no effective disease-modifying drug has been developed due to the disease's complicated chronic pathology, affecting not only cartilage but also subchondral bone and synovium3.

Mesenchymal stem cells (MSCs), first described in human bone marrow4, are emerging as promising cell-based therapeutics for a wide range of diseases5. MSCs are also isolated from synovium6, and synovial MSCs are particularly suited for cartilage repair as they

http://dx.doi.org/10.1016/j.joca.2015.12.018

1063-4584/© 2016 The Authors. Published by Elsevier Ltd and Osteoarthritis Research Society International. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

N. Ozeki et al. / Osteoarthritis and Cartilage xxx (2016) 1—10

have superior proliferation7 and chondrogenic potential8. We have previously reported on synovial MSCs for cartilage regeneration in an osteochondral defect model in rabbits9 and pigs10. We also obtained satisfactory results by administration of synovial MSCs to cartilage defects in humans11.

Recently, several studies have shown the effectiveness of a single intra-articular injection of stem cells for OA in animals12-15 and humans16-18. The definitive mechanisms behind stem cell-based injection therapies are still unclear, but putative mechanisms have been proposed such as anti-inflammatory effects13, regeneration of the meniscus12, or direct differentiation into functional chondrocytes14. The number of injected stem cells has been shown to decrease immediately following a single dose injec-tion13,19, which indicate that a single treatment is not enough to overcome the long-term pathology of chronic OA. In this study, we compared the chondroprotective effects of periodic injections of synovial MSCs with that of a single injection, and analyzed cell kinetics and behavior with rat luciferase, LacZ, and GFP expressing synovial MSCs in a rat OA model. We also determined MSCs' trophic factor contributions by species-specific microarray and PCR analyses using human synovial MSCs in a rat OA model.

Materials and methods

Animals

A total of 76 wild type male Lewis rats (Charles River Laboratories Japan, Kanagawa, Japan) at 10-12 weeks old were used for the experiments. All animal care and experiments were conducted in accordance with the institutional guidelines of the Animal Committee of Tokyo Medical and Dental University. Luciferase expressing transgenic rats (N = 4)20, LacZ expressing transgenic rats (N = 4) and green fluorescence protein expressing transgenic rats (N = 4)21 (all provided byJichi Medical University, Tochigi, Japan) were also used for analyses of in vivo imaging, detection of X-Gal staining, and flow cytometry. Rats were anesthetized by isoflurane inhalation and intraperitoneal injection of tribromoethanol.

Preparation of synovial MSCs

This study was approved by an institutional review board, and informed consent was obtained from all human subjects. To prepare human synovial MSCs, synovium was harvested from donors during anterior cruciate ligament (ACL) reconstruction surgery for ligament injury. Synovial MSCs from rats or humans were prepared as previously described8,22-24. In summary, harvested synovium was minced and digested with collagenase for three hours, and cells were disseminated into complete culture medium and incubated at 37° C with 5% humidified CO2. After 14 days, cells were collected and preserved at -80°C. For an injection, 1 x 106 synovial MSCs were prepared in 50 ml of phosphate buffered saline (PBS). Synovial MSCs from luciferase, LacZ, and GFP expressing rats were also prepared in the same manner (Luc+ MSCs, LacZ+ MSCs and GFP+ MSCs).

Surgery and injection of synovial MSCs

After the patellar tendon was dislocated laterally, ACL was transected completely. The rats were allowed to walk freely in their cages. From 1week following the surgery, the rats had intra-articular injection of PBS alone (control group) every week, 1 x 106 synovial MSCs at one time (single group), or every week (weekly group).

Evaluations of cartilage degeneration

Both the femoral condyle and the tibial plateau were evaluated by India Ink staining for macroscopic observation. For the histo-logical examination, both femoral and tibial cartilage were fixed in 4% paraformaldehyde for 7 days, and decalcified in 20% ethylene diamine tetra acetic acid (EDTA) solution for 21 days, then embedded in paraffin wax. The specimens were sectioned in the sagittal plane at 5 mm and stained with safranin-o and fast green. Cartilage degeneration was evaluated using the Osteoarthritis Research Society International (OARSI) scoring system in the medial part of the femur and tibia25.

In vivo bioluminescent imaging

A noninvasive bioimaging system IVIS (Xenogen, Alameda, CA) was used after single or weekly injections of 1 x 106 Luc+ MSCs. Under anesthesia with isoflurane, d-luciferin was administrated (20 mg/ml, 50 ml) at 1, 7,14, 21, and 28 days after the first injection of Luc+ MSCs, and photons were detected by IVIS (N = 3, each). As a normal control, intact knees were also evaluated. The signal intensity was quantified as photon flux in units of photons per seconds in the region of interest.

Detection of LacZ expression

X-Gal staining was performed after injection of 1 x 106 LacZ+ MSCs. The rats were sacrificed at 1 day after injection, and the knee specimens were fixed with a fixative solution (0.2% glutaraldehyde, 2 mM MgCl2, and 5 mM EDTA) in PBS for 30 min at room temperature and rinsed 3 times in PBS to wash out the fixative solution. They were treated with an X-gal staining solution (1 mg/ml 5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside, 2 mM MgCl2, 6 mM potassium ferricyanide, and 6 mM potassium ferrocyanide) under incubation at 37° C for 3 h. After taking pictures of macroscopic findings, they were subsequently fixed again in 4% para-formaldehyde. The specimens were decalcified with 0.5 M EDTA (pH 7.5) for 21 days, and embedded in paraffin wax, then sectioned and counterstained with eosin.

Flow cytometry

1 x 106 GFP+ MSCs were injected 1week after the ACLT, then synovium was harvested 1day after the injection, following digestion with collagenase V for three hours. After filtering through a 70 mm cell strainer and centrifuging at 1500 rpm for 5 min, cells were suspended in ice-cold Hank's balances salt solution and then stained for 30 min on ice with a monoclonal antibody of APC-conjugated CD90 (Becton, Dickinson and Company, Franklin Lakes, NJ, USA). Synovial fluid was also extracted from the same knee, and prepared in the same manner without collagenase digestion. Propidium iodide (PI) fluorescence was measured, and a live cell gate was defined that excluded the cells positive for PI. Additional gates were defined as positive for GFP and CD90. Double positive cells were further analyzed for PE-conjugated CD29, PE-conjugated CD31, and PEcy7-conjugated CD45 (Biolegend, San Diego, CA, USA). Flow-cytometric analysis and sorting were performed on a MoFlo (Beckman Coulter, Brea, CA, USA), and the data were analyzed using FlowJo software (Tree Star, Ashland, OR, USA)26.

Oligonucleotide microarray

RNA was isolated from synovium 1, 3, and 7 days after injection of 1 x 106 hMSCs (n = 4, each) using TRIzol solution (Invitrogen Life

N. Ozeki et al. / Osteoarthritis and Cartilage xxx (2016) 1—10

Technologies, Carlsbad, CA, USA) and High Pure RNA Isolation Kit (Roche Applied Sciences, Indianapolis, IN, USA). A microarray analysis was performed using 500 ng of total RNA from each sample and GeneChip® Rat Genome 230 2.0 probe arrays (Affymetrix, Santa Clara, CA, USA) and/or GeneChip® Human U-133 plus 2.0 probe arrays. Data was analyzed with GeneSpring GX software version 12.5 (Agilent Technologies, Palo Alto, CA, USA). To normalize the variations in staining intensity among chips, the signal values for all genes on a given chip were divided by the median value for the expression of all genes on the chip. To eliminate genes containing only background signals, genes were selected only if the raw values of the signal were more than the lower limit of the confidence interval, and expression of the gene was judged to be 'present' using MAS5 algorithm in Expression Console Version1.1 (Affymetrix). The microarray data were deposited in the Gene Expression Omnibus (GEO accession no. GSE61617).

Quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR)

First-strand cDNAs were synthesized using a Transcriptor Firststrand cDNA synthesis kit (Roche), and Q-PCR analyses were performed using a LightCycler 480 Probe Master system (Roche) with human-specific b-actin primers and a probe. For the assay of human and rat mRNA, PCR primers of human and rat RNA are listed in Supplementary Tables 2 and 3. Relative mRNA expression levels were calculated as described by Niikura et al.27.

Statistical analysis

Statistical analysis was performed with the StatView 5.0 program (SAS Institute, Cary, NC). Comparisons between the two groups for PCR analysis were analyzed using the Mann—Whitney's U-test. Comparisons between multiple groups were analyzed using the Kruskal—Wallis test. P values less than 0.05 were considered to be statistically significant. Error bars indicated 95% CI.

Results

Weekly intra-articular injections of synovial MSCs inhibit OA progression

To determine the chondroprotective effects of synovial MSCs in detail, we administered weekly intra-articular injections of synovial MSCs in a rat ACLT model to compare these effects against those of a single injection [Fig. 1(A)]. In the control group (PBS injection) or single injection group, significant cartilage lesions appeared at 8 weeks macroscopically [Fig. 1(B) and (C); white arrows] and histologically [Fig. 1(D) and (F)]. These cartilage pathologies progressively worsened at 12 weeks. On the other hand, weekly injections of synovial MSCs better preserved cartilage both macroscopically and histologically, when stained with safranin-o. The OARSI score in the weekly injection group was significantly better in the medial tibial plateau at 8 and 12 weeks [Fig. 1(E)] and in the medial femoral condyle at 12 weeks [Fig. 1(G)]. Cartilage immunostaining for type II collagen was also better preserved in the weekly injection group (Supplementary Figs. 1 and 2). To assess synovial inflammation, synovium including the infrapatellar fat pad of the whole knee joint was evaluated. In the control group and the single injection group at 8 and 12 weeks, hyperplasia of the synovial lining layers and/or increased cell infiltration were/was observed [Supplementary Fig. 3(A); black arrows]. Synovitis score at 12 weeks was lower in the weekly injection group than in the

control and single injection groups in all three rats we examined in each group [Supplementary Fig. 3(B)].

Weekly injections of synovial MSCs maintain viable cells in knees

To examine cell viability in vivo, we evaluated photons from Luc+ MSCs using an IVIS system. In vitro imaging of luciferase activity showed that the system enabled detection of as few as one thousand MSCs over the background in the linear dose-dependent output of luminescence [Fig. 2(A)]. For in vivo imaging analyses in the weekly group, we injected Luc+ MSCs just after the evaluation of IVIS, except the initial injection [Fig. 2(B)]. We evaluated three rats per group at 5 time points. Although synovial MSCs rapidly decreased at 1 week and almost disappeared at 2—3 weeks after a single injection in the ACLT knee, MSCs could be detected at every evaluation time point in the case of weekly injections in all cases [Fig. 2(C) and (D)].

To examine the migration of MSCs in the knee joint, we then evaluated X-Gal staining after the injection of LacZ+ MSCs [Fig. 3(A)]. One day post injection, dark blue areas of LacZ+ MSCs were present in the synovium both macroscopically and histolog-ically [Fig. 3(B); red arrow, and C; black arrows]; however, no migration of LacZ+ MSCs into the cartilage or meniscus was observed. To determine if the injected MSCs were able to survive in the synovial fluid, we cultured synovial fluid 1 day after the injection of LacZ+ MSCs into the ACLT knee, and colonies were evaluated by staining for X-Gal and crystal violet [Supplementary Fig. 3(A)]. We analyzed synovial fluid from three knees, and colonies were barely detectable from the intact knee. On the other hand, a constant number of colonies were detected after ACLT knees irrespective of MSC injection [Supplementary Fig. 4(A) and (B)]. Approximately 10% of colonies were stained with X-Gal [Fig. 4(A)], which indicated a small number of injected synovial MSCs survived in the synovial fluid.

Injected synovial MSCs retain their MSCs properties after migration into the synovium

We next determined if the injected cells would retain their undifferentiated features by use of GFP+ MSCs [Fig. 4(A)]. Using flow cytometry, we sorted GFP+ MSCs engrafted within the synovium [Fig. 4(B)] and synovial fluid from four knees. Before the injection, most GFP+ MSCs expressed CD29 and CD90, and lacked the expression of CD31 and CD45 [Fig. 4(C)]. After migration within the synovium, the surface markers of the GFP+ cells did not change [Fig. 4(D) and Supplementary Fig. 5]. The ratio of GFP+ cells against total live cells gradually decreased with time [Fig. 4(E) and (F)], but the ratio of CD90 positive cells within the GFP+ cells was maintained at approximately 90% even at 28 days after injection [Fig. 4(G)]. From the synovial fluid, the ratio of GFP+ cells against total live cells was also low [Fig. 4(E) and (F)] and their CD90 positive ratio was approximately 90% [Fig. 4(G)]. The sorted GFP+ cells differentiated into chondrocytes, adipocytes, and were calcified in vitro (Supplementary Fig. 6).

Injected synovial MSCs increase expressions of genes related to chondroprotection and anti-inflammation after migration into the synovium

To quantify the number of synovial MSCs engrafted within the synovium, we applied a xenotransplantation model19,28 and prepared standard curves of b-actin from synovium mixed with predetermined numbers of hMSCs just before homogenization [Fig. 5(A)]. We also purified RNA from the synovium injected with hMSCs, and assayed it with a quantitative RT-PCR assay specific for

N. Ozeki et al. / Osteoarthritis and Cartilage xxx (2016) 1—10

Fig. 1. Weekly intra-articular injections of synovial MSCs inhibited OA progression and attenuated synovitis in a rat OA model. (A) Schema of the study. (B) Representative macroscopic features of the tibial plateau from three specimens. Cartilage erosion is indicated by white arrows. Scale bar, 1 mm. A, anterior; P, posterior; M, medial; L, lateral. (C) Representative histological sections stained with safranin-o for the tibial plateau. Left panel, low magnification. Scale bar, 1 mm. OARSI score for each section is indicated. Right panel, high magnification. Scale bar, 100 mm. (D) OARSI score for histology of the tibial plateau (N = 6, P < 0.05). Error bars represent 95% confidence intervals. (E) Representative features of infrapatellar fat pad stained with hematoxylin & eosin. Proliferation of lining layer or infiltrations of cells are indicated with black arrows. Left panel, low magnification. Scale bar, 500 mm. Right panel, high magnification. Scale bar, 100 mm. (F) Synovitis score for histology. Each score is plotted (N = 3). (G) OARSI score for histology of the femoral condyle (N = 6, P < 0.05). Error bars represent 95% confidence intervals.

human b-actin. After injection of 106 hMSCs, approximately 104 hMSCs were recovered in the rat synovium at 1 day, and 103 hMSCs were recovered at 3 days [Fig. 5(B) and (C)]. No human MSCs were detected at 7 days.

The use of a xenotransplantation made it possible to follow changes in both the injected hMSCs and the endogenous rat cells in the synovium simultaneously. We evaluated the human and rat transcriptomes in the synovium injected with 106 hMSCs 1 day after injection. Rat synovium mixed with 104 hMSCs was used as a control. Fold changes of human b-actin and other housekeeping genes, such as hGAPDH, hNPAT, and hCTNNBl, were between 0.5 and 2.0, suggesting that the normalization is applicable (Supplementary

Table 4). After filtering for cross-hybridization with human mRNA, the data showed that after migration within the synovium, the human mRNAs on day 1 for 5 genes increased over 100-fold, 21 genes increased over 50-fold, and about 255 genes increased over 10-fold, and about 1060 genes increased over 2-fold. The 20 most highly up-regulated human transcripts included hPRG-4 and hBMP-2 (Table I). We then confirmed these significant increases by RT-PCR for hPRG-4, hBMP-2, and hBMP-6, which are related to cartilage homeostasis, and hTSG-6, a known anti-inflammatory cytokine [Fig. 5(D)]. These results strongly suggest that various growth factors were secreted by hMSCs after migration into the synovium. With regard to transcripts related to chondrogenesis, hCol2a1

N. Ozeki et al. / Osteoarthritis and Cartilage xxx (2016) 1—10

Fig. 2. Weekly injections of synovial MSCs maintained viable cells in knees. (A) In vitro bioluminescent imaging of varying numbers of luciferase positive synovial MSCs. (B) Schema for the in vivo imaging analysis. In the weekly group, the injection was performed immediately after the analysis at 1, 2, and 3 weeks. (C) In vivo imaging analyses after injection of Luc+ MSCs into the knee joint (N = 3, each). (D) Sequential quantification of luminescence intensity. Each intensity is plotted and the average is shown as a line graph (N = 3).

mRNA increased by only 1.4 fold and hSox9 mRNA was not detected, which suggested that hMSCs showed no signs of chondrogenesis in the synovium (Supplementary Table 5). Microarray data also indicated that injection of human MSCs up-regulated the expression of 406 rat transcripts 2-fold or more (Supplementary Table 6). We focused on the pro-inflammatory cytokines or cytokines related to proliferation such as rIL-1@ and rVEGF, but no significant difference was observed by real-time RT-PCR [Fig. 5(D)].

Discussion

Not single but weekly intra-articular injections of synovial MSCs inhibited OA progression and attenuated synovitis in a rat OA model. After the administration of MSCs into the knee joint, it is well known that the number of cells rapidly decreases13,19, and we obtained similar results from our experiments. These suggest that periodic injections are necessary to maintain both the number and the efficacy of MSCs for the long term to improve the condition of OA. In fact, a single injection of synovial MSCs had a minimal effect in the current study, which demonstrates the importance of

periodic administration of stem cells against OA. This is the first study demonstrating the effectiveness of periodic MSC dosing for chronic pathology in the knee joint.

There have been reports describing the effect of a single injection of MSCs in different OA models12—15. Ter Huurne et al. injected adipose-derived stem cells into the knee in a mouse OA model induced by intra-articular injection of collagenase13. OA progression was inhibited when evaluated 42 days after a single injection of stem cells due to the attenuation of synovitis. However, although GFP-transfected cells were detectable in the synovium 24 h after injection, they were no longer detectable at day 5 and thereafter. Murphy et al. induced OA in goats by removal of the medial meniscus and resection of the ACL, followed by intra-articular injection of bone marrow MSCs. Meniscus regeneration was stimulated though the ACL was not repaired, and progression of the articular cartilage degeneration was reduced at 20 weeks12. In this model, GFP-transfected MSCs were still detected in the center of neomeniscal tissue 6 weeks after injection. They concluded that the chondroprotective effects of stem cells were due to the regeneration of the meniscus. These reports of short vs long-term stem cell

N. Ozeki et al. / Osteoarthritis and Cartilage xxx (2016) 1—10

Fig. 3. Injected synovial MSCs migrated into the synovium or suspended in synovial fluid. (A) Schema for the study using LacZ+ MSCs. (B) Macroscopic features of LacZ+ MSCs migrated into the synovium after X-Gal staining. (N = 3, Scale bar, 1 mm, S; synovium, F; femoral condyle, T; tibial cartilage, M; meniscus) (C) Histological sections counterstained with eosin (Scale bars, 500 mm/low magnification, 20 mm/high magnification). LacZ+ MSCs are indicated by black arrows. The boxed areas in insets are shown at a higher-magnification in the larger panels. (D) X-Gal staining and colony forming assay for synovial fluid of normal knee, ACLT knee, and ACLT knee injected with LacZ+ MSCs 1 day before (N = 3, each). In "ACLT knee injected with LacZ+ MSCs," the same dish was shown before and after crystal violet staining. Scale bar, 100 mm. (E) Quantification of colony number. Each value is plotted and the average is shown as a crossbar (N = 3).

survival and migration are often contradictory, even though OA protection is observed in each. Here, we found that stem cells remained primarily in the synovium after repeated injections, though they disappeared 7 days after a single injection. We therefore, administered subsequent dosings accordingly, every 7 days. It is unclear if these multiple injections would improve the therapeutic effects seen in some single injection models where long-term cell survival is observed.

From our analyses using LacZ+ or GFP+ MSCs, injected MSCs primarily migrated into the synovium, but they were not detected in the cartilage. This is a distinct result from previous studies in which stem cells survived within the osteochondral defect exposed to bone marrow29,30. We transplanted synovial MSCs 1 week after ACLT, and at that time cartilage degeneration was not obvious. In cases where MSCs were administrated later, when the articular cartilage was more degenerated, MSCs may be detected in the cartilage.

Other efforts have been directed at investigating an endogenous synovial MSC niche, and Kurth etal. suggested that stem cells in the synovium were distinct from pericytes from the results of double nucleoside labeling analysis and immunohistochemistry31. We previously identified that the numbers of a-smooth muscle actin-positive vessels and CD31 + endothelial cells were different among the harvest sites of the synovium in human OA, and these numbers were strongly correlated with the number of colony-forming synovial MSCs32. We speculated that the niche of these cells would be near vascular regions. However, the behavior of MSCs after transplantation remain uncertain33. When administered MSCs differentiate into the injured tissue, they lose their MSC properties. To determine this, we injected GFP+ MSCs into the wild

type rat knee and sorted only GFP+ cells by flow cytometry that migrated into the synovium. We surprisingly identified that almost all GFP+ cells retained their MSCs properties, positive for CD90 and CD29, negative for CD31, CD45. In addition, their properties remained even at 4 weeks after the injection, although the number of GFP+ cells decreased. From these findings, we confirmed that synovial MSCs maintained viability with stemness after engraft-ment in the synovium, without being directed to differentiation. This is the first study elucidating that MSCs retained stemness after intra-articular injection.

Underlying mechanisms of stem cell therapy include two major roles: the replacement of injured tissues34, and the production of trophic factors35. To clarify the effect of synovial MSCs after migration, we analyzed rat synovium with its migrated hMSCs by species-specific microarrays and PCR analyses. hMSCs gene expression changed after hMSCs migrated within the rat synovium. We found significant up-regulation of several cytokines, such as PRG-4, BMPs, and TSG-6, which are key trophic factors for chon-droprotection. PRG-4, known as lubricin, normally produced by synoviocytes or superficial zone chondrocytes, plays an important role in homeostasis and maintenance of cartilage36,37. Intra-articular injection of lubricin prevented cartilage degeneration in a rat ACLT model38. BMP-2 and -6 are critical for the differentiation of chondrocytes, cartilage matrix synthesis39, and cartilage pro-tection40. TSG-6 has been previously reported to be secreted by engrafted MSCs to suppress inflammation in myocardial infarction, peritonitis, and inflammatory cornea injury models28,41,42. Two major pathologies of OA include degeneration of articular cartilage and synovitis1; therefore, PRG-4 and BMPs chondroprotectively affected the articular cartilage, and TSG-6 delayed secondary

N. Ozeki et al. / Osteoarthritis and Cartilage xxx (2016) 1—10

Fig. 4. Injected synovial MSCs retained their MSC properties after migration into the synovium. (A) Schema for the flow-cytometric assay. PI (B) Macroscopic features of the GFP+ MSC injected knee. The boxed area is shown at a higher-magnification. Scale bar, 80 mm (C) Flow-cytometric profiles of GFP+ MSCs before injection. (D) Flow-cytometric profiles of GFP+ MSCs 1 day after migration within the synovium. (E) Representative flow-cytometric profiles of cells derived from the synovium and in the synovial fluid for GFP+ and CD90 positive cells. In the negative control, synovial cells without any GFP+ MSCs were analyzed without CD90 antibody (N = 4, each). (F) Sequential ratio of GFP+ MSCs per total alive cells in the synovium. Each value is plotted and the average is shown as a crossbar (N = 4). (G) Ratio of CD90 positive cells in the GFP+ cells (N = 4).

cartilage degeneration through attenuating synovitis. We considered that these multiple trophic effects have a considerable advantage in the use of stem cells, and these advantages are not accomplished by the administration of one single drug.

The analyses for synovial MSC viability and gene expression were performed in a xenotransplantation model. Previous reports clarified the function of MSCs using human cells into mice28 or rats43. The migrated MSCs may endure a different response from the joint environment and elicit a different response to the joint in the case of an allogeneic transplantation model. Therefore, more attention should be given to the possible differences between allogeneic or autologous transplantation model.

We did not identify the trophic effect of the MSCs that survived in the synovial fluid as was done in the synovium because the amount of synovial fluid was extremely low, and the number of surviving cells in the synovial fluid was small. Previously, we reported that higher numbers of colonies were obtained from synovial MSCs of ACL injured knee, meniscus injured knee, or OA knees than healthy control knees44-46. These results indicated that stem cells are recruited to the synovial fluid as a response to injury of the joint, and they may have some biological effect. It is the next step to analyze what induces the recruitment of stem cells into the joint environment.

In this model, we used young rats with open growth plates; however, the population of OA patients is usually older adults. In human studies, we reported that the proliferation and differentiation potentials of synovial MSCs in elderly OA patients were comparable to those in young patients with ACL injury47. But, though the quality of isolated synovial MSCs from donors of various ages may be similar; we also have to consider that the joint environment in which we apply synovial MSCs is critically affected by age, degree of OA, and condition of inflammation.

To achieve repeated injections, the preparation of a considerable number of stem cells is necessary. Autologous synovial MSCs are preferable to allogeneic MSCs to avoid immune reactions in clinical situations. From our experience, a sufficient number of MSCs can be prepared from the synovium in most donors7,11 and yields, proliferation and chondrogenic potential of synovial MSCs were similar in young donors and elderly donors47. Therefore, it is possible to obtain a high number of synovial MSCs for the repeated injections in OA patients. However, the optimal number of stem cells required for an intra-articular injection is still controversial. Jo et al. performed 3 dose-escalation cohorts in OA patients; a low-dose (1 x 107 cells), mid-dose (5 x 107) and high-dose (1 x 108) group. Second-look arthroscopy and histology showed that the

N. Ozeki et al. / Osteoarthritis and Cartilage xxx (2016) 1—10

Fig. 5. Injected synovial MSCs increased expression of genes related to chondroprotection and anti-inflammation after migration into the synovium. (A) Schema for the xenotransplantation assay. (B) Standard curve showing relationship between the number of hMSCs mixed with rat synovium and DCt (difference between expression levels of human and rat b-actin). DCt from the rat synovium after injection of hMSCs was also plotted. (C) The number of viable hMSCs in rat synovium after injection. Each value was obtained from the standard curve shown in Fig. 5(B). The value at 7 days was below the detection limit. (D) Human specific and rat specific gene expressions in the synovium of rat at 1 day by RT-PCR. Control group had 104 hMSCs mixed with synovium. (E) Possible mechanism delaying the progression of cartilage degeneration by weekly injections of synovial MSCs in a rat OA model. Injected synovial MSCs migrate into the synovium and express BMPs, PRG-4, TIMP-1, etc. that prevent cartilage degradation, and also TSG-6 that inhibits inflammation.

Table I

The top 20 human transcripts up-regulated in the hMSCs that migrated within the synovium

Probe set ID Gene symbol Gene title FC

209278_s_at TFP12 Tissue factor pathway inhibitor 2 252.6

206007_at PRG4 Proteoglycan 4 162.3

206300_s_at PTHLH Parathyroid hormone-like hormone 130.5

1558048_x_at T.L Transcribed locus 107.5

219043_s_at LOC285359///PDCL3 Phosducin-like 3 pseudogene///phosducin-like 3 102.6

210145_at PLA2G4A Phospholipase A2, group IVA (cytosolic, calcium-dependent) 92.5

205289_at BMP2 Bone morphogenetic protein 2 87.4

201909_at RPS4Y1 Ribosomal protein S4, Y-linked 1 75.5

210108_at CACNA1D Calcium channel, voltage-dependent, L type, alpha 1D subunit 63.6

203477_at COL15A1 Collagen, type XV, alpha 1 63.3

208051_s_at PA1P1 Poly(A) binding protein interacting protein 1 62.8

205932_s_at MSX1 msh homeobox 1 61.6

203840_at BLZF1 Basic leucine zipper nuclear factor 1 61.6

237496_at T.L Transcribed locus 61.0

229174_at C3orf38 Chromosome 3 open reading frame 38 59.4

202196_s_at DKK3 dickkopf homolog 3 (Xenopus laevis) 57.2

229088_at ENPP1 Ectonucleotide pyrophosphatase/phosphodiesterase 1 56.5

232458_at COL3A1 Collagen, type III, alpha 1 56.0

217790_s_at SSR3 Signal sequence receptor, gamma (translocon-associated protein gamma) 55.5

219163_at ZNF562 zinc finger protein 562 53.2

RNA was recovered 1 day after the intra-articular injection of human MSCs for analysis on human specific microarrays (Affymetrix). Signal intensities were compared between samples from synovium 1 day after injection of the ACLT knees and control samples from 104 hMSCs added to the synovium of ACLT knees. Values are fold increase over values for control. T.L; Transcribed locus. Almost all the flags in these control samples were 'Absent'.

N. Ozeki et al. / Osteoarthritis and Cartilage xxx (2016) 1—10

high dose group had a significant reduction of cartilage defects and improvement in function with reduced pain17. However, Kim et al. prepared an average of 4.3 x 106 stem cells for OA patients48, and saw significant improvement of both IKDC and Tegner activity scores. Ongoing studies in the field, like these, will help identify the optimal number of synovial MSCs and frequency of injections for clinical use in OA patients.

Finally, we summarized the chondroprotective mechanism of MSC injection in the current model. Without any treatment after the ACLT, cartilage degeneration appears at the cartilage surface and gradually progresses into the deep zone, demonstrating chronic OA. When synovial MSCs are injected into the knee joint, most of them migrate into the synovium and surviving cells maintained their MSC properties without differentiating into another lineage. Then they produce PRG-4 and BMPs for cartilage homeostasis and TSG-6 for anti-inflammation [Fig. 5(E)]. These factors contribute to chondroprotection and prevent the progression of OA, but migrated MSCs do not survive in the long term, and the number of MSCs rapidly decreases after the injection. Therefore a single administration of synovial MSCs had only a temporary and a minimal effect against the chronic pathology in OA. When synovial MSCs are injected weekly, the number of MSCs and the chondroprotective effect is maintained in the long term, which confirms reliable effects in inhibiting OA progression.

Author contributions

NO: Conception and design. Collection, analysis, and interpretation of the data. Drafting of the article. TM: Critical revision of the article for important intellectual content. HK: Interpretation of data. YN: Interpretation of data. MU: Collection of data. RS: Collection of data. KY: Collection of data. MM: Collection and analysis of data. KT: Interpretation of data. YM: Collection and analysis of data. CA: Interpretation of data. EK: Interpretation of data. KM: Collection, analysis, and interpretation of data. KF: Collection, analysis, and interpretation of data. TS: Interpretation of data. IS: Conception and design, final approval of the article.

Conflict of interest

Eiji Kobayashi has been a medical adviser and received honorarium from Berthold Japan K.K. since 2014. Other authors have no conflict of interest.

Acknowledgments

We would like to thank Ms Miyoko Ojima for her expert help with this study. All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published.

This study was supported by the Highway Program for Realization of Regenerative Medicine from Japan Science and Technology Agency (JST) and Japan Agency for Medical Research and Development (AMED).

Supplementary data

Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.joca.2015.12.018.

References

1. Abramson SB, Attur M. Developments in the scientific understanding of osteoarthritis. Arthritis Res Ther 2009;11:227.

2. Bijlsma JW, Berenbaum F, Lafeber FP. Osteoarthritis: an update with relevance for clinical practice. Lancet 2011;377:2115-26.

3. Sellam J, Berenbaum F. The role of synovitis in pathophysiology and clinical symptoms of osteoarthritis. Nat Rev Rheumatol 2010;6:625-35.

4. Friedenstein AJ, Deriglasova UF, Kulagina NN, Panasuk AF, Rudakowa SF, Luria EA, et al. Precursors for fibroblasts in different populations of hematopoietic cells as detected by the in vitro colony assay method. Exp Hematol 1974;2:83-92.

5. Kebriaei P, Isola L, Bahceci E, Holland K, Rowley S, McGuirk J, et al. Adult human mesenchymal stem cells added to cortico-steroid therapy for the treatment of acute graft-versus-host disease. Biol Blood Marrow Transplant 2009;15:804-11.

6. De Bari C, Dell'Accio F, Tylzanowski P, Luyten FP. Multipotent mesenchymal stem cells from adult human synovial membrane. Arthritis Rheum 2001;44:1928-42.

7. Nimura A, Muneta T, Koga H, Mochizuki T, Suzuki K, Makino H, et al. Increased proliferation of human synovial mesenchymal stem cells with autologous human serum: comparisons with bone marrow mesenchymal stem cells and with fetal bovine serum. Arthritis Rheum 2008;58:501-10.

8. Sakaguchi Y, Sekiya I, Yagishita K, Muneta T. Comparison of human stem cells derived from various mesenchymal tissues: superiority of synovium as a cell source. Arthritis Rheum 2005;52:2521-9.

9. Koga H, Muneta T, Nagase T, Nimura A, Ju YJ, Mochizuki T, et al. Comparison of mesenchymal tissues-derived stem cells for in vivo chondrogenesis: suitable conditions for cell therapy of cartilage defects in rabbit. Cell Tissue Res 2008;333:207-15.

10. Nakamura T, Sekiya I, Muneta T, Hatsushika D, Horie M, Tsuji K, et al. Arthroscopic, histological and MRI analyses of cartilage repair after a minimally invasive method of transplantation of allogeneic synovial mesenchymal stromal cells into cartilage defects in pigs. Cytotherapy 2012;14:327-38.

11. Sekiya I, Muneta T, Horie M, Koga H. Arthroscopic transplantation of synovial stem cells improves clinical outcomes in knees with cartilage defects. Clin Orthop Relat Res 2015;473: 2316-26.

12. Murphy JM, Fink DJ, Hunziker EB, Barry FP. Stem cell therapy in a caprine model of osteoarthritis. Arthritis Rheum 2003;48: 3464-74.

13. ter Huurne M, Schelbergen R, Blattes R, Blom A, de Munter W, Grevers LC, et al. Antiinflammatory and chondroprotective effects of intraarticular injection of adipose-derived stem cells in experimental osteoarthritis. Arthritis Rheum 2012;64: 3604-13.

14. Sato M, Uchida K, Nakajima H, Miyazaki T, Guerrero AR, Watanabe S, et al. Direct transplantation of mesenchymal stem cells into the knee joints of Hartley strain guinea pigs with spontaneous osteoarthritis. Arthritis Res Ther 2012;14:R31.

15. Toghraie FS, Chenari N, Gholipour MA, Faghih Z, Torabinejad S, Dehghani S, et al. Treatment of osteoarthritis with infrapatellar fat pad derived mesenchymal stem cells in rabbit. Knee 2011;18:71-5.

16. Koh YG, Choi YJ, Kwon SK, Kim YS, Yeo JE. Clinical results and second-look arthroscopic findings after treatment with adipose-derived stem cells for knee osteoarthritis. Knee Surg Sports Traumatol Arthrosc 2015;23:1308-16.

17. Jo CH, Lee YG, Shin WH, Kim H, Chai JW, Jeong EC, et al. Intra-articular injection of mesenchymal stem cells for the treatment of osteoarthritis of the knee: a proof-of-concept clinical trial. Stem Cells 2014;32:1254-66.

19. Horie M, Choi H, Lee RH, Reger RL, Ylostalo J, Muneta T, et al. Intra-articular injection of human mesenchymal stem cells (MSCs) promote rat meniscal regeneration by being activated to express Indian hedgehog that enhances expression of type II collagen. Osteoarthritis Cartilage 2012;20:1197-207.

N. Ozeki et al. / Osteoarthritis and Cartilage xxx (2016) 1—10

20. Hakamata Y, Murakami T, Kobayashi E. "Firefly rats" as an organ/cellular source for long-term in vivo bioluminescent imaging. Transplantation 2006;81:1179-84.

21. Inoue H, Ohsawa 1, Murakami T, Kimura A, Hakamata Y, Sato Y, et al. Development of new inbred transgenic strains of rats with LacZ or GFP. Biochem Biophys Res Commun 2005;329: 288—95.

22. Yoshimura H, Muneta T, Nimura A, Yokoyama A, Koga H, Sekiya 1. Comparison of rat mesenchymal stem cells derived from bone marrow, synovium, periosteum, adipose tissue, and muscle. Cell Tissue Res 2007;327:449—62.

23. Okuno M, Muneta T, Koga H, Ozeki N, Nakagawa Y, Tsuji K, et al. Meniscus regeneration by syngeneic, minor mismatched, and major mismatched transplantation of synovial mesen-chymal stem cells in a rat model. J Orthop Res 2014;32: 928—36.

24. Ozeki N, Muneta T, Matsuta S, Koga H, Nakagawa Y, Mizuno M, et al. Synovial mesenchymal stem cells promote meniscus regeneration augmented by an autologous achilles tendon graft in a rat partial meniscus defect model. Stem Cells 2015;33:1927—38.

25. Pritzker KP, Gay S, Jimenez SA, Ostergaard K, Pelletier JP, Revell PA, et al. Osteoarthritis cartilage histopathology: grading and staging. Osteoarthritis Cartilage 2006;14:13—29.

26. Mabuchi Y, Morikawa S, Harada S, Niibe K, Suzuki S, Renault-Mihara F, et al. LNGFR(+)THY-1(+)VCAM-1(hi+) cells reveal functionally distinct subpopulations in mesenchymal stem cells. Stem Cell Rep 2013;1:152—65.

27. Niikura T, Hak DJ, Reddi AH. Global gene profiling reveals a downregulation of BMP gene expression in experimental atrophic nonunions compared to standard healing fractures. J Orthop Res 2006;24:1463—71.

28. Lee RH, Pulin AA, Seo MJ, Kota DJ, Ylostalo J, Larson BL, et al. 1ntravenous hMSCs improve myocardial infarction in mice because cells embolized in lung are activated to secrete the anti-inflammatory protein TSG-6. Cell Stem Cell 2009;5: 54—63.

29. Koga H, Shimaya M, Muneta T, Nimura A, Morito T, Hayashi M, et al. Local adherent technique for transplanting mesenchymal stem cells as a potential treatment of cartilage defect. Arthritis Res Ther 2008;10:R84.

30. Mizuno M, Kobayashi S, Takebe T, Kan H, Yabuki Y, Matsuzaki T, et al. Brief report: reconstruction of joint hyaline cartilage by autologous progenitor cells derived from ear elastic cartilage. Stem Cells 2014;32:816—21.

31. Kurth TB, Dell'accio F, Crouch V, Augello A, Sharpe PT, De Bari C. Functional mesenchymal stem cell niches in adult mouse knee joint synovium in vivo. Arthritis Rheum 2011;63: 1289—300.

32. Nagase T, Muneta T, Ju YJ, Hara K, Morito T, Koga H, et al. Analysis of the chondrogenic potential of human synovial stem cells according to harvest site and culture parameters in knees with medial compartment osteoarthritis. Arthritis Rheum 2008;58:1389—98.

33. Bianco P, Cao X, Frenette PS, Mao JJ, Robey PG, Simmons PJ, et al. The meaning, the sense and the significance: translating the science of mesenchymal stem cells into medicine. Nat Med 2013;19:35—42.

34. O'Sullivan J, D'Arcy S, Barry FP, Murphy JM, Coleman CM. Mesenchymal chondroprogenitor cell origin and therapeutic potential. Stem Cell Res Ther 20H;2:8.

35. Caplan AI, Dennis JE. Mesenchymal stem cells as trophic mediators. J Cell Biochem 2006;98:1076-84.

36. Rhee DK, Marcelino J, Baker M, Gong Y, Smits P, Lefebvre V, et al. The secreted glycoprotein lubricin protects cartilage surfaces and inhibits synovial cell overgrowth. J Clin Invest 2005;115:622-31.

37. Flannery CR, Zollner R, Corcoran C, Jones AR, Root A, Rivera-Bermudez MA, et al. Prevention of cartilage degeneration in a rat model of osteoarthritis by intraarticular treatment with recombinant lubricin. Arthritis Rheum 2009;60:840-7.

38. Jay GD, Torres JR, Warman ML, Laderer MC, Breuer KS. The role of lubricin in the mechanical behavior of synovial fluid. Proc Natl Acad Sci USA 2007;104:6194-9.

39. Sekiya I, Colter DC, Prockop DJ. BMP-6 enhances chondro-genesis in a subpopulation of human marrow stromal cells. Biochem Biophys Res Commun 2001;284:411-8.

40. Blaney Davidson EN, Vitters EL, van Lent PL, van de Loo FA, van den Berg WB, van der Kraan PM. Elevated extracellular matrix production and degradation upon bone morphogenetic protein-2 (BMP-2) stimulation point toward a role for BMP-2 in cartilage repair and remodeling. Arthritis Res Ther 2007;9: R102.

41. Choi H, Lee RH, Bazhanov N, Oh JY, Prockop DJ. Anti-inflammatory protein TSG-6 secreted by activated MSCs attenuates zymosan-induced mouse peritonitis by decreasing TLR2/NF-kB signaling in resident macrophages. Blood 2011;118:330-8.

42. OhJY, Roddy GW, Choi H, Lee RH, Ylostalo JH, RosaJr RH, et al. Anti-inflammatory protein TSG-6 reduces inflammatory damage to the cornea following chemical and mechanical injury. Proc Natl Acad Sci USA 2010;107:16875-80.

43. Horie M, Driscoll MD, Sampson HW, Sekiya I, Caroom CT, Prockop DJ, et al. Implantation of allogenic synovial stem cells promotes meniscal regeneration in a rabbit meniscal defect model. J Bone Joint Surg Am 2012;94:701-12.

44. Morito T, Muneta T, Hara K, Ju YJ, Mochizuki T, Makino H, et al. Synovial fluid-derived mesenchymal stem cells increase after intra-articular ligament injury in humans. Rheumatology (Oxford) 2008;47:1137-43.

45. Matsukura Y, Muneta T, Tsuji K, Koga H, Sekiya I. Mesenchymal stem cells in synovial fluid increase after meniscus injury. Clin Orthop Relat Res 2014;472:1357-64.

46. Sekiya I, Ojima M, Suzuki S, Yamaga M, Horie M, Koga H, et al. Human mesenchymal stem cells in synovial fluid increase in the knee with degenerated cartilage and osteoarthritis. J Orthop Res 2012;30:943-9.

47. Mochizuki T, Muneta T, Sakaguchi Y, Nimura A, Yokoyama A, Koga H, et al. Higher chondrogenic potential of fibrous syno-vium- and adipose synovium-derived cells compared with subcutaneous fat-derived cells: distinguishing properties of mesenchymal stem cells in humans. Arthritis Rheum 2006;54: 843-53.

48. Kim YS, Choi YJ, Koh YG. Mesenchymal stem cell implantation in knee osteoarthritis: an assessment of the factors influencing clinical outcomes. Am J Sports Med 2015;43:2293-301.