TISSUE-SPECIFIC STEM CELLS

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1 TISSUE-SPECIFIC STEM CELLS In Vivo Identification and Induction of Articular Cartilage Stem Cells by Inhibiting NF-jB Signaling in Osteoarthritis a The Key Laboratory of Stem Cell Biology, Institute of Health Sciences, Shanghai Jiao Tong University School of Medicine (SJTUSM) & Shanghai Institutes for Biological Sciences (SIBS), Chinese Academy of Sciences (CAS), Shanghai, People s Republic of China; b Department of Orthopaedics and Traumatology, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong SAR, People s Republic of China; c Shanghai Key Laboratory of Orthopaedic Implant, Department of Orthopaedics, Shanghai Ninth People s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, People s Republic of China; d Orthopaedics Research Laboratory, Research Center, Sacre-Coeur Hospital, University of Montreal, Montreal, Quebec, Canada; e Cell and Molecular Biology Laboratory, Department of Orthopaedics, Alpert Medical School of Brown University/ Rhode Island Hospital, Providence, Rhode Island, USA Correspondence: Xiaoling Zhang, Ph.D., Room 1230 (116 Mail Box), Life Science Building Block A, Institute of Health Sciences, 320 Yueyang Road, Xuhui District , Shanghai, People s Republic of China. Telephone: ; Fax: ; xlzhang@sibs.ac.cn Received January 30, 2015; accepted for publication June 30, 2015; first published online in STEM CELLS EXPRESS August 3, VC AlphaMed Press /2014/$30.00/ /stem.2124 WENXUE TONG, a,b YIYUN GENG, a YAN HUANG, a,c YU SHI, a SHENGNAN XIANG, a NING ZHANG, a LING QIN, b QIN SHI, d QIAN CHEN, e KERONG DAI, a,c XIAOLING ZHANG a,c Key Words. Articular cartilage stem cells Identification Induction NF-jB signaling Osteoarthritis ABSTRACT Osteoarthritis (OA) is a highly prevalent and debilitating joint disorder characterized by the degeneration of articular cartilage. However, no effective medical therapy has been found yet for such condition. In this study, we directly confirmed the existence of articular cartilage stem cells (ACSCs) in vivo and in situ for the first time both in normal and OA articular cartilage, and explored their chondrogenesis in Interleukin-1b (IL-1b) induced inflammation environment and disclose whether the inhibition of NF-jB signaling can induce ACSCs activation thus improve the progression of experimental OA. We found an interesting phenomenon that ACSCs were activated and exhibited a transient proliferative response in early OA as an initial attempt for selfrepair. During the in vitro mechanism study, we discovered IL-1b can efficiently activate the NFjB pathway and potently impair the responsiveness of ACSCs, whereas the NF-jB pathway inhibitor rescued the ACSCs chondrogenesis. The final in vivo experiments further confirmed ACSCs activation were maintained by NF-jB pathway inhibitor, which induced cartilage regeneration, and protected articular cartilage from injury in an OA animal model. Our results provided in vivo evidence of the presence of ACSCs, and disclosed their action in the early OA stage and gradual quiet as OA process, presented a potential mechanism for both cartilage intrinsic repair and its final degradation, and demonstrated the feasibility of inducing endogenous adult tissuespecific mesenchymal stem cells for articular cartilage repair and OA therapy. STEM CELLS 2015;33: SIGNIFICANCE STATEMENT The study confirmed the existence of articular cartilage stem cells (ACSCs) in vivo and in situ for the first time both in normal and OA articular cartilage, found an interesting phenomenon that ACSCs were activated and exhibited a transient proliferative response in early OA as an initial attempt for self-repair, confirmed ACSCs activation were maintained by NF-jB pathway inhibitor, which induced cartilage regeneration, and protected articular cartilage from injury in an OA animal model, demonstrated the feasibility of inducing endogenous adult tissue-specific mesenchymal stem cells for articular cartilage repair and OA therapy. INTRODUCTION Osteoarthritis (OA) is a common joint disease characterized by the degeneration of articular cartilage. However, no effective medical therapy has been found for such condition. This condition is considered as the consequence of an imbalance between anabolism and catabolism of the articular cartilage, particularly by an increase in catabolism. In early OA, cells react with a transient proliferative response (clonal growth). This reaction increases the synthesis of cartilage matrix, including types II, IX, and XI collagens, aggrecan, pericellular type IV collagen, and anabolic factors including bone morphogenetic protein-2 (BMP-2), insulin-like growth factor-1, BMP-7, and inhibin b-a/activin (members of the TGF-b superfamily), as an early attempt at repair. However, this endeavor cannot overcome all catabolic processes [1 4]. However, as OA progresses, inflammatory cytokines, such as interleukin-1 (IL-1), are believed to elevate in the catabolic process, which play crucial roles in OA progression [5, 6]. The cellular response in OA is complex, and the more information becomes available, the more complex it seems. Thus, an understanding of the cellular processes that STEM CELLS 2015;33: VC AlphaMed Press 2015

2 3126 In Vivo Identification and Induction of ACSCs regulate the functional activities of cells in the cartilage tissue in both physiological and pathological conditions is essential for the development of more effective strategies for treating patients with OA and altering the natural history of this disorder. The presence of mesenchymal stem cells (MSCs) is a key component for a rapid and successful regeneration of various tissues [7]. MSCs are clonogenic populations with the capacity for extensive self-renewal and selective differentiation into various mesenchymal tissue lineages [8]. Cells with MSC features have been found in several human adult tissues and organs. However, the articular cartilage appears to be hypocellular and avascular depending on the diffusion for its nutrient delivery. Several studies had demonstrated that the articular cartilage consists only terminally differentiated cells and lacks MSCs in adults [7]. However, this doctrine has been challenged in recent years by several studies, which demonstrated that a MSCs population resides in the superficial zone (SZ) of the articular cartilage [9 13]. According to several reports, articular cartilage MSCs are significantly increased in OA cartilage [11 13]. The increased activation of Notch, Stro- 1, vascular cell adhesion molecule-1, and Sox9 in OA cartilage can indicate a regeneration response signaling in cartilage [7]. These results provide evidence for the hypothesis that cartilage has the ability to self-repair and articular cartilage stem cells (ACSCs) were involved in this biological behavior, and provide a new view for OA and cartilage regeneration research. Nevertheless, no in situ studies have identified that ACSCs were activated in OA and elucidated the signaling pathway may be involved during this process. 5-Bromo-2-deoxyuridine (BrdU) has already been established for in vivo stem cell labeling in many tissues [14 17], BrdU is incorporated into the DNA when the cell undergoes mitosis and can thereafter be detected by antibodies against BrdU. Thus, BrdU labeling can be applied for detecting slow cycling cells (e.g., MSCs) because the rapidly proliferating cells will lose their incorporated BrdU at early time points due to the gradual dilution during mitosis. Ki67, a cell cycling protein expressed in proliferating cells, can be used as a marker for detecting MSC activation (under proliferating) within a tissue [18, 19]. This study is the first to test the location of ACSCs and to determine whether ACSCs was active in OA using BrdU and Ki67 two-color immunofluorescence. Only BrdU is detectable when ACSCs are still in a resting state; whereas both BrdU and Ki67 are detectable when ACSCs are activated (under proliferating condition). Tissue MSCs are usually in a resting state until they are activated by certain signals, for example, growth or repair of an injury within the tissue or a nearby tissue [20 23]. In this study, we hypothesized that cartilage trauma stimulates ACSCs responsiveness to change from a resting state to an active state (cell proliferation), thus inducing self-repair. This study, which provides in vivo evidence of the presence of ACSCs and the ability of the cartilage s self-repair, aims to determine whether ACSCs were activated during the regeneration response in OA. We also evaluated whether ACSCs responsiveness is impaired by increased inflammatory signals of OA. Considering that IL-1b plays a major role in cartilage catabolism, we assessed the mechanism of IL-1b signal on ACSCs proliferation and differentiation, evaluated the proliferation response of ACSCs during the progression of experimental VC AlphaMed Press 2015 OA and whether inhibiting NF-jB signaling could induce ACSCs and suspend the progression of experimental OA, and deduced a new possible strategy for future OA treatment of cartilage injuries targeting ACSCs for cartilage regeneration. MATERIALS AND METHODS Labeling and Detection of ACSCs In Vivo We intraperitoneally injected BrdU (Sigma, 50 lg per g b.wt.) into 2-day-old newborn Sprague-Dawley (SD) rats twice daily for 3 days. The BrdU-labeled cells were then detected on the frozen sections using the BrdU antibody (Sigma-Aldrich, B2531), and the mitosis of the cells was further investigated by immunofluorescence using the Ki67 antibody (Abcam, ab16667). For immunofluorescent staining, we incubated the sections with primary antibodies to BrdU and Ki67 (the dilution ratio was 1:200) overnight at 48C. For P65 antibody (Santa Cruz, sc-33039) in the following cellular immunofluorescent experiment, the dilution was 1:300. Secondary antibodies (DyLight 488, A21202; DyLight 594, A21207, Invitrogen, Carlsbad, CA) conjugated with fluorescence were added on the dilution of 1:500 to the sections, and slides were incubated in the dark at room temperature for 1 hour. Subsequently, the sections were microphotographed for histomorphometric measurements on the entire cartilage area (LEICA TCSSP5, Wetzlar, Germany). The percentage of Ki67 and BrdU positive (Ki671/ BrdU1) cells in BrdU positive (BrdU1) cells in the entire cartilage area per specimen were assessed in five sequential sections per sample in each group by two independent observers who were blinded to the experimental groups. OA Animal Model and Histology Animal handling and experimental procedures were performed following the approval from the Institute of Health Sciences Institutional Animal Care and Use Committee. The 8- week-old ACSCs labeled SD rats (200 g) were randomized into four groups [OA group, sham group, BAY treatment group and dimethyl sulfoxide (DMSO) group]. OA was induced by medial collateral ligament (MCL) transection and medial meniscal tear of the knee joints, as previously described [24]. Briefly, the animals were anesthetized, and surgery was performed to transect the MCL. The medial meniscus was cut through the full thickness to induce the joint destabilization of the right knee (OA group). Sham animals underwent the same surgical procedure without any ligament transection or meniscal tear as the control group (sham group). After surgery, each rat was given penicillin once a day for the first 3 days. For the NF-jB pathway inhibitor treatment (BAY treatment group), the BAY (10 lm), or the equivalent volume of vehicle (DMSO, DMSO group), was injected intra-articularly directly into the knee joints of recipient rats 1 day after the surgery (10 ll per joint per rat twice a week).- The operated rats were euthanized on 0, 2, 8, 14, 30, 60, or 90 days after surgery (n per group), and the samples of the knee joints were collected and fixed in acetone for 30 minutes, followed by a decalcifying step in 4% EDTA for at least 1 month. Fluid was changed every 2 days and embedded in optimal cutting temperature (O.C.T., Sakura 4583, Japan). The 4-lm-thick sagittal-oriented sections of the knee STEM CELLS

3 Tong, Geng, Huang et al joint medial compartment were processed for hematoxylin eosin, safranin O, and fast green staining. The Osteoarthritis Research Society (OARSI) scores were then calculated by a single observer who was blinded to the experimental group as previously described [25]. Cell Isolation and Culture Previous studies used differential adhesion to fibronectin in vitro to identify epidermal MSCs and articular cartilage MSCs [10, 26, 27]. Fibronectin is expressed in developing mammalian articular cartilage, in addition to the classic fibronectin receptor integrin subunits a5 and b1 [28]. Thus, we used fibronectin in an in vitro adhesion assay to identify ACSCs. Cartilage slices were harvested from the articular cartilages of SD rats (12 weeks old) hip and knee joints and then cut into small pieces. The pieces were subsequently washed with sterile phosphate buffered saline (PBS, ph 7.4) and digested in trypsin-edta (0.25%, wt/vol; Invitrogen) for 10 minutes and collagenase type II (0.02%, wt/vol; Sigma-Aldrich, St. Louis, MO, dissolved in serum free 1/1 Dulbecco s modified Eagle s medium (DMEM)/F-12, SH B, HyClone, Logan, Utah) for 5 hours. The isolated cells were seeded into six-well plates (Corning, Corning, NY) coated with 10 lg/ml of fibronectin (R&D Systems, Minneapolis, MN) in 1:1 DMEM/F-12 medium with 10% fetal bovine serum, 50 U/ml penicillin, and 50 mg/ ml streptomycin. The DMEM/F-12 medium was removed after 20 minutes, and the adhered cells (ACSCs) were digested in trypsin-edta again and recultured in10 cm dishes (Corning) in DMEM/F-12 medium. The cells left over in the removed medium (chondrocytes) cultured in 10 cm dishes with the same medium as ACSCs. For the isolation of bone marrow-derived mesenchymal stem cells (BM-MSCs), SD rats heparinized bone marrow aspirates were diluted with a-minimum essential medium (a- MEM, SH B, HyClone, Logan, Utah) and centrifuged. The mononuclear cell pellet was then resuspended in complete a-mem and cultured. The medium was replaced after 24 hours and every 3 days thereafter. The osteogenesis, adipogenesis, and chondrogenesis ability of ACSCs and BM-MSCs were assessed as previously described [29, 30]. Flow Cytometry After the cells (ACSCs and chondrocytes, respectively) harvest and preparation of single-cell suspension, the cells were stained for 45 minutes with or without (negative control) antibodies. The cells were then washed three times in PBS and resuspended. The cells were subjected to fluorescenceactivated cell sorting using a FACSCAN program (BD Biosciences, San Diego, CA) with DIVA software. The antibodies used included anti-cd45 (557015; BD Biosciences), anti-cd34 (sc- 7324; Santa Cruz Biotechnology, Santa Cruz, CA), anti-cd31 (555027; BD Biosciences), anti-cd44 (ab33900; Abcam, Cambridge, U.K.), and anti-cd90.1 (551401; BD Biosciences). These antibodies were replaced with PE- or FITC-conjugated isotypematched IgG1 in the negative controls (IC002P or IC002F, R&D Systems, Inc.). Triplicates of cells from three rats were examined in this assay. Aggregate Chondrogenesis Model The ACSCs were suspended and added to a polypropylene V-bottom 96-well plate ( cells/well) (Corning). The plate was then spun at 400g for 5 minutes. The supernatant was replaced by chondrogenesis low-glucose Dulbecco s modified Eagle s medium (LG-DMEM, SH B, HyClone, Logan, Utah) containing l-glutamine and sodium pyruvate with 1% ITS 1 (ITS) premix, 40 lg/ml of proline, 100 nm of dexamethasone, 50 lg/ml of ascorbic acid 2-phosphate (all from Sigma-Aldrich), and 10 ng/ml of Recombinant Human TGF-b1 (100-21, PeproTech). 10 ng/ml Recombinant Human Interleukin-1b (IL-1b) (200-01B, PeproTech) was added as inflammatory stimulus. The chondrogenesis LG-DMEM medium was changed every other day [31]. The cells were cultured for 7 weeks to form large, compacted pellets. Pictures of the aggregates were taken after 7 weeks in culture. The images were analyzed using ImageJ software (NIH Image, Bethesda, MD) to measure the diameter of the aggregates. Real-Time Quantitative PCR Total RNA was extracted from cartilage pellets after 7 weeks culture (three per group) after homogenized and lysed by high speed homogenizer in TriPure reagent (Roche Applied Science, Mannheim, Germany). The concentration and purity of mrna were detected by Nano-Drop 2000 (Thermo Fisher Scientific, MA). Real-time PCR was performed after reverse transcription reaction using a Roche LC 480 system with SYBR Premix (TaKaRa, Inc., Dalian, People s Republic of China). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control. Data were analyzed by comparison Ct (2- DDCt) method, and expressed as fold change compared with GAPDH. The primer sequences are shown in Supporting Information Table S1. Each sample was analyzed in triplicate. Immunoblotting The total protein extractions complied with the Roche Applied Science TriPure Isolation Reagent Instruction Manual. Briefly, ethanol (100%, 0.3 ml/1 ml TriPure) was added to the interphase and organic phases left after the RNA extraction. The samples were then incubated for 3 minutes at 208C and centrifuged at 2,000g for 5 minutes at 48C. The phenol ethanol supernatant was removed to a new tube and used to isolate the protein. Isopropanol (1.5 ml/1 ml TriPure) was then added to the supernatant and mixed by inverting several times, followed by incubation for 10 minutes at 208C. The sample was centrifuged at 12,000g for 10 minutes at 48C, and the supernatant was discarded. Each protein pellet was resuspended with 1 ml 0.3 M of guanidine hydrochloride in 95% ethanol for three times, washed once with 100% ethanol, and dissolved by adding 1% sodium dodecyl sulfate (SDS). The SDS-pellet mixture was then incubated at 508C to completely solubilize the protein. Proteins were fractionated by SDS-polyacrylamide gel electrophoresis after the addition of 5 3 loading buffer, boiled for 3 minutes. After 40 minutes running under 100 V (10 minutes) and 120 V (30 minutes), the fractionated proteins were transferred to a nitrocellulose membrane under 275 ma for 100 minutes and then detected using the anti- GAPDH (G9545, Sigma-Aldrich), anti-smad7 (sc-11392, Santa Cruz, CA), and antitransforming growth factor TGF-bRII (sc- 400, Santa Cruz, CA). Immunoreactive bands were quantitatively analyzed in triplicate by normalizing the band intensities to their respective controls on scanned films with Quantity One software. VC AlphaMed Press 2015

4 3128 In Vivo Identification and Induction of ACSCs Figure 1. In vivo identification of articular cartilage stem cells (ACSCs). (A, B): BrdU and Ki67 were both positive in nearly all cells in the new born rats. (C): Only stem cells that retained the BrdU label (BrdU1 ACSCs) were detected in adult rats (8-week-old), whereas no Ki67 expression was detected in ACSCs. Indirect Immunofluorescence and Confocal Laser Microscopy ACSCs were grown on glass coverslips. The cells were left untreated or pretreated with BAY for 1 hour, followed by stimulation with IL-1b (10 ng/ml) for 20 minutes. The cells were then fixed with 4% PBS-paraformaldehyde for 15 minutes and P65 antibody (Santa Cruz, sc-33039) was diluted at 1:300, followed by the secondary antibody (DyLight 488, A21202, Invitrogen, Carlsbad, CA) on the dilution of 1:500. Process for immunofluorescence as previously described [29]. Statistical Analysis Results were expressed as mean 6 standard deviation. Statistical analysis was carried out using one-way ANOVA with Tukey s multiple comparisons test or two-way ANOVA with Sidak s multiple comparisons test, and paired-sample t test (for animal experiment in Fig. 7 only). p values of less than.05,.01, or.001 were considered statistically significant, very significant, or extremely significant, respectively. RESULTS In Vivo Identification of ACSCs The BrdU labeling effect was confirmed on the frozen sections by BrdU and Ki67 immunofluorescence immediately after the intraperitoneally injection. Since nearly all the cells in the newborn rats were in an actively proliferative statue, BrdU could bind to the genome of all the cells, and Ki67 was widely expressed. So BrdU and Ki67 were both positive by immunofluorescence technique. As mentioned in the introduction, after the rats grow up (after 8-week-old), only stem cells that retained the BrdU label (BrdU 1 cells, ACSCs) could be detected, whereas no Ki67 expression was detected in ACSCs VC AlphaMed Press 2015 (Fig. 1), indicating that ACSCs were existent and not activated (resting state) at normal cartilage. ACSCs Were Activated in the Context of OA With the use of BrdU and Ki67 immunofluorescence, we found that only the BrdU 1 ACSCs were detected on the cartilage in the sham (control) group, no Ki67 expression was detected in ACSCs (Fig. 2A). In OA group, Ki67 was gradually detected in ACSCs as the OA progressed, and the percentage of Ki671/BrdU 1 ACSCs in BrdU 1 ACSCs peaked at approximately 65% (Fig. 2B 2D, 2H) on the 14th day after the OAinducing surgical operation. During these days, cartilage degeneration gradually progressed, as indicated by the increase in OARSI grade (Fig. 2I). This phenomenon suggested a transient amplification of ACSCs (activated state) in early OA. Moreover, as cartilage degeneration became more severe (Day 30, Day 60, and Day 90) and OA grades further increased (Fig. 2I), a significantly reduced number of transient proliferating Ki671/BrdU 1 ACSCs were visualized (Fig. 2E 2G, 2H). Notably, on the 90th day, they were scarcely detected (Fig. 2G). This phenomenon suggested the ACSCs were activated in early OA, but the active ACSCs loss gradually as the development of OA progresses. The highest frequencies of Ki671/BrdU1 ACSCs were found in the superficial zone (SZ) of the cartilage, as shown in Figure 2. The BrdU1 ACSCs (ACSCs in resting state) and the Ki671-only cells (general somatic cells under mitosis) in the samples (Supporting Information Fig. S1) were observed at each time points, and the appearance of these cells showed an irregular feature. The BrdU1 cells in the deep zone of the cartilage at the Day 60 time point (Supporting Information Fig. S1) may suggest another source of stem cells in OA which has been reported by several reports [12]. STEM CELLS

5 Tong, Geng, Huang et al Figure 2. Articular cartilage stem cells (ACSCs) were activated in the context of osteoarthritis (OA). (A): BrdU1 ACSCs were detected on the cartilage of the sham group, but no Ki67 expression was observed in ACSCs. (B D, H): In OA group, Ki67 was gradually detected in BrdU1 ACSCs as OA progressed and maximized at 14 days after the OA-inducing surgical operation. (E G, H): The number of transient proliferating ACSCs (Ki671/BrdU1 ACSCs) visualized as cartilage degeneration significantly decreased. (A G, I): The cartilage degeneration and The Osteoarthritis Research Society grade showed the OA development. Cyan nuclear represent BrdU1 ACSCs and white nuclear represent Ki671/BrdU1 ACSCs. *, p <.05; **, p <.01; ***, p <.001 between two groups. Isolation and Verification of Multidirectional Differentiation Potential of ACSCs ACSCs were isolated from the rat articular cartilage through the fibronectin-adhesion method. Immunofluorescence was used to test BrdU immediately after the cell isolation using fibronectin-coated culture dishes (Fig. 3A), the result showed that 86.2% 6 7.4% cells were BrdU-retaining cells. The chondrocytes (the cells left over after the fibronectin- VC AlphaMed Press 2015

6 3130 In Vivo Identification and Induction of ACSCs Figure 3. Isolation and multidirectional differentiation potential verification of ACSCs. (A): Two different magnifications showed that about 86.2% 6 7.4% cells isolated by fibronectin were BrdU-retaining cells. (B): Chondrocytes scarcely expressed CD44, CD90, CD31, CD34, and CD45. (C): ACSCs highly expressed CD44 and CD90, but scarcely expressed CD31, CD34, and CD45. (D): The colony-forming efficiency of ACSCs was confirmed at low density 12 days after plating. (E G): Osteogenesis [evaluated by (E) ALP quantification assay and (F) alizarin red staining] and adipogenesis [evaluated by (G) oil red O staining] capabilities of the ACSCs were significantly greater than chondrocytes, but weaker than BM-MSCs. (H, I): The chondrogenesis [evaluated by (H) alcian blue staining and (I) diameter of the micromass] capability of the ACSCs was significantly greater than BM-MSCs. Cho2 denotes general chondrocytes with general medium; Cho 1 denotes general chondrocytes with differentiated medium; ACSCs 1 denotes ACSCs with differentiated medium; and BMSC 1 denotes BM-MSCs with differentiated medium. The values are the mean and SEM of triplicate experiments. **, p <.01; ***, p <.001 between two groups. Abbreviations: ACSC, articular cartilage stem cell; ALP, alkaline phosphatase; BMSC, bone marrow derived mesenchymal stem cells. conglutination) and ACSCs surface markers were detected by flow cytometry analysis. The chondrocytes showed a low expression of Thy-1/CD90 (a glycosylphosphatidylinositolanchored glycoprotein) (6.4%) and hyaluronan receptor (CD44) (5.6%) (Fig. 3B), whereas ACSCs showed a high expression of CD90 (>95%) and CD44 (>70%) (Fig. 3C). CD90 and CD44 are both MSC surface markers. Hematopoietic and endothelial markers, such as CD31, CD34, and CD45, were scarcely expressed both in chondrocytes and ACSCs (Fig. 3B, 3C). Figure 3D shows the colony-forming efficiency of ACSCs. Colonies were defined as a cluster of more than 32 cells representing a population of cells derived from more than five population doublings of a single cell. The osteogenesis and adipogenesis differentiation capability of ACSCs were much greater than that of chondrocytes (the cells left over after the fibronectin-conglutination), but weaker than that of the BM- MSCs (Fig. 3E 3G). However, the chondrogenesis capability of VC AlphaMed Press 2015 ACSCs was greater than that of the BM-MSCs, as shown by the alcian blue staining (Fig. 3H) and the micromass diameter (Fig. 3I). This phenomenon suggests that ACSCs have a pluralpotent differential potential, but are lopsided and prone to chondrogenesis. IL-1b Suppressed the TGF-b1-Induced Chondrogenesis in Rat ACSCs To examine the effect of growth factors and proinflammatory cytokines on ACSCs differentiation, ACSCs chondrogenesis was induced by TGF-b1, and the role of IL-1b in this process was investigated. The size of the cell aggregates substantially increased after 7 weeks when stimulated with TGF-b1 (10 ng/ ml) in the pellet culture system; however, the pellets were distinctly smaller when costimulated with IL-1b (0.1, 1, and 10 ng/ml) (Fig. 4A 4C). The gene expressions of four cartilage markers (SOX9, COL IIa, aggrecan, and Has 2) were quantified STEM CELLS

7 Tong, Geng, Huang et al Figure 4. IL-1b suppressed the TGF-b1 induced chondrogenesis in rat articular cartilage stem cells. (A): Dose dependence of the IL-1binhibited effects on aggregates cultured with increasing concentrations of IL-1b in the presence and absence of TGF-b1, which was also demonstrated by (B) pellet diameter. (C): Toluidine blue staining. (D): qpcr analysis of the four cartilage markers, namely, SOX9, COL IIa, aggrecan, and Has 2. The values are the mean and SEM of triplicate experiments. *, p <.05; ***, p <.001 between two groups. Abbreviation: GAPDH, glyceraldehyde 3-phosphate dehydrogenase. by quantitative PCR (qpcr). The levels of marker gene expression obtained in the TGF-b1-treated group were greater than those in the control (no TGF-b1) group and costimulated group with IL-1b (Fig. 4D). These results suggested that the proinflammatory cytokines IL-1b inhibited the TGF-b1-induced ACSCs chondrogenesis by suppressing the expression of the transcription factor (SOX9), extracellular matrix (ECM) (COL IIa and aggrecan), and related synthetase (hyaluronansynthetase 2), and these inhibitory effects on ACSCs chondrogenesis were dose-dependent. NF-jB Pathway Plays an Essential Role in IL-1b- Induced Inhibition of ACSCs Chondrogenesis The expression of several important factors was detected in the TGF-b/Smad pathway to obtain the key factors during the TGF-b1-induced and IL-1b-suppressed ACSCs chondrogenesis process. Plasminogen activator inhibitor 1 (PAI-1), a gene that perfectly reflects the activation of the TGF-b/Smad pathway, was significantly stimulated by TGF-b1 and suppressed by IL- 1b in a dose-dependent manner (Fig. 5A). During these processes, IL-1b did not have any effect on the expression of Smad2, Smad3, Smad4, and TGF-bRI induced by TGF-b1 (Supporting Information Fig. S2). However, Smad7 and TGF-bRII expressions were obviously inversely influenced by IL-1b (Fig. 5B 5E). Given that IL-1b exerts several of its effects by the NF-jB pathway, we investigated the involvement of the NFjB pathway in the IL-1b regulation of TGF-bRII and Smad7. Nearly all p65 was transported into the nucleus of the cells when ACSCs were stimulated with 10 ng/ml of IL-1b (Fig. 5F); IL-1b-induced p65 transport was inhibited by BAY (a widely used NF-jB pathway inhibitor) in a dose-dependent manner. When BAY concentration reached 10 lm, p65 was scarcely transported into the nucleus. Another index, MMP13, which is a gene rapidly induced by the NF-jB pathway, was intensely induced by the treatment of 10 ng/ml of IL-1b, but was suppressed by BAY in a dosedependent manner (Fig. 5G). NF-jB Pathway Inhibitor Could Restore TGF-b1- Induced Chondrogenesis Inhibited by IL-1b Pellet culture experiments were implemented with a combination of 10 ng/ml of TGF-b1, 10 ng/ml of IL-1b, and three concentrations of BAY (1, 5, and 10 lm). Gross observations (Fig. 6A) and pellet diameters (Fig. 6B) indicate that 10 ng/ml of TGF-b1 can significantly induce ACSCs chondrogenesis, whereas IL-1b (10 ng/ml) suppressed this process. BAY can restore TGF-b1-induced chondrogenesis inhibited by IL-1b in a dose-dependent manner. The cartilage marker gene SOX9, COL IIa, aggrecan, and Has 2 expression was detected by qpcr, the results further confirmed the functions of TGF-b1 and IL-1b and the dose-dependent restorative effect of BAY (Fig. 6C). The level of PAI-1 suppressed by IL-1b was restored by NF-jB pathway inhibitor BAY in a dose-dependent manner (Fig. 6D). Smad2, Smad3, Smad4, and TGF-bRI (Supporting Information Fig. S3) were upregulated by TGF-b1, but were not regulated by IL-1b and BAY , the effect of IL-1b on Smad7 and TGF-bRII could be reversed by BAY (Fig. 6E, 6F). The expression of VC AlphaMed Press 2015

8 3132 In Vivo Identification and Induction of ACSCs Figure 5. NF-jB pathway has a key role in IL-1b-induced inhibition of articular cartilage stem cells chondrogenesis. (A): PAI-1 was significantly stimulated by TGF-b1 and suppressed by IL-1b in a dose-dependent manner. (B): TGF-b1 downregulated the Smad7 expression, whereas IL-1b significantly reversed the effect of TGF-b1 in a dose-dependent manner. Notably, 10 ng/ml of IL-1b completely offset 10 ng/ml of TGF-b1 suppression on Smad7 and significantly increased the Smad7 expression compared with the control (no TGF-b1) group (approximately 1.4-fold) and the TGF-b1 treatment-only group (approximately 2.6-fold). (C): Conversely, TGF-bRII was significantly induced by TGF-b1 and suppressed by IL-1b in a dose-dependent manner. The results are expressed as the percentage of controls after normalization against GAPDH. (D): Western blot results of Smad7 and TGF-bRII showed the same trend as that of qpcr results. (E): The statistics of the Western blot results. (F, G): The NF-jB pathway was inhibited by BAY (F): The suppression of p65 transport into the nucleus induced by IL-1b. (G): MMP13 downregulation. The values are the mean and SEM of triplicate experiments. *, p <.05; **, p <.01; ***, p <.001 between two groups. Abbreviation: GAPDH, glyceraldehyde 3-phosphate dehydrogenase. Smad7 and TGF-bRII at the protein level exhibited the same trend as the qpcr results (Fig. 6G, 6H). Inhibition of NF-jB Signaling Can Induce ACSCs Activation and Retard the Progression of Experimental OA The inhibition function of the NF-jB signaling with BAY by inducing ACSCs activation was further tested through an OA animal model. The percentage of Ki671/ BrdU1 ACSCs in BrdU1 ACSCs in the DMSO (control) group and BAY treatment group was gradually increased during the early OA progression (Fig. 7A, 7B, 7E, 7F, 7I). The percentage of Ki671/BrdU1 ACSCs in BrdU1 ACSCs in the DMSO group peaked by approximately 60% on the 14th day (Fig. 7B, 7I), whereas the percentage of Ki671/BrdU1 ACSCs in BrdU1 ACSCs in the BAY treatment group peaked VC AlphaMed Press 2015 by approximately 75% on the 30th day (Fig. 7G, 7I). The Ki671/BrdU1 ACSCs in the DMSO group (after 14 days postoperation) and in the BAY treatment group (after 30 days postoperation) gradually reduced with the progression of experimental OA (Fig. 7B 7D, 7G 7J). However, the percentage of Ki671/BrdU1 ACSCs in BrdU1 ACSCs in the BAY group was approximately 25% on the 90th day, in contrast to 7% in the DMSO group. Therefore, an increased percentage of transient amplifying Ki671/BrdU1 ACSCs occurred in the BAY treatment group. Moreover, cartilage degeneration and OA progression (Fig. 7J) were inhibited in the BAY treatment group compared with the DMSO group. These data denote that the NF-jB pathway is crucial for regulating ACSCs activity and for blocking the NF-jB pathway to induce ACSCs activation, which may be a possible method for OA therapy. STEM CELLS

9 Tong, Geng, Huang et al Figure 6. NF-jB pathway inhibitor restored TGF-b1-induced chondrogenesis inhibited by IL-1b. (A C): BAY distinctly restored TGF-b1-induced chondrogenesis inhibited by IL-1b in a dose-dependent manner. (A): Naked eye pellet size, (B) pellet diameter, and (C) qpcr analysis of SOX9, COL IIa, aggrecan, and Has 2. (D): The level of PAI-1 suppressed by IL-1b, and restored by BAY in a dosedependent manner. (E): TGFb-1 downregulated Smad7 expression and IL-1b markedly reversed the effect of TGF-b1. However, the IL-1b effect could be reversed by BAY (F): Conversely, TGF-bRII was significantly induced by TGF-b1, suppressed by IL-1b, and restored by BAY in a dose-dependent manner. (G, H): Western blot results of Smad7 and TGF-bRII, which showed the same trend as that of the qpcr results. (H): The statistics of the Western blot results. The values are the mean and SEM of triplicate experiments. *, p <.05; **, p <.01; ***, p <.001 between two groups. Abbreviation: GAPDH, glyceraldehyde 3-phosphate dehydrogenase. DISCUSSION The current challenge is to obtain more information regarding the spontaneous cartilage repair mechanisms to develop new regenerative therapies for the treatment of OA. Mesenchymal cells are involved in the regeneration processes that accompany cartilage tissue degeneration. After enzymatic digestion of healthy cartilage [10] and OA tissue [11 13], cells with mesenchymal characteristics are defined with the help of surface markers to demonstrate that MSCs are present in adult human articular cartilage, and their frequency is increased in the OA cartilage [11, 13]. This observation has VC AlphaMed Press 2015

10 3134 In Vivo Identification and Induction of ACSCs Figure 7. Inhibition of NF-jB signaling induced articular cartilage stem cells (ACSCs) activation and retarded the progression of experimental osteoarthritis (OA). (A I): The BrdU1 ACSCs were gradually activated (Ki671/BrdU1 ACSCs) from the resting state in early OA, maximized in about the 14th day (A, B) in the DMSO group and the 30th day (E G) in the BAY treatment group after the OAinducing surgical operation. (B D, G J): The Ki671/BrdU1 ACSCs in the DMSO group (after 14 days postoperation) and in the BAY treatment group (after 30 days postoperation) gradually reduced with the progression of OA. The Ki671/BrdU1 ACSCs maintained approximately 25% on the 90th day in the BAY group (H, I) in contrast to 7% in the DMSO group (D, I). (A H, J): The cartilage degeneration and The Osteoarthritis Research Society grade were more improved in the BAY treatment group compared with the DMSO group. Cyan nuclear represent BrdU1 ACSCs and white nuclear represent Ki671/BrdU1 ACSCs. ***, p <.001 between two groups. implications for understanding the intrinsic repair capacity of articular cartilage and raises the possibility that these MSCs may be involved in the pathogenesis of arthritis. We have VC AlphaMed Press 2015 limited knowledge of the spontaneous cartilage repair and no in vivo report about ACSCs by now. Tissue stem cells are very challenging research field for many years, and many STEM CELLS

11 Tong, Geng, Huang et al ponderable researches have been reported on adiposederived stem cells, tendon-derived stem cells, muscle-derived stem cells, and so forth. However, there are rarely reports about cartilage-derived stem cells, especially their function in vivo. In this study, we directly confirmed the existence of ACSCs in vivo and in situ for the first time both in normal and OA articular cartilage, discovered that ACSCs were activated from resting state in OA. Thus, our results provide in vivo evidence regarding the ability of articular cartilage to self-repair, this ability probably dependent on the presence of ACSCs as the number of transient proliferating ACSCs was synchronous with the OA progression in early stage. This study showed that the highest frequencies of ACSCs harbor in the SZ of cartilage, it is consistent with the special growth mechanism named appositional growth strongly speculated the ACSCs, if existed, should locate in the SZ of cartilage [9]. Considering the abundant and relatively sclerous ECM in cartilage and its avascular character, stem cells of other source are difficult to migrate to the SZ of cartilage. However, with the vascularization during the late OA and the porous character of the subchondral bone, the chondrogenic progenitor cells were found in deep zone cartilage in late OA may migrate from the subchondral bone or even the bone marrow [12]. Because of the lack of unique and well-known marker for ACSCs, the in vivo migration research of ACSCs is limited in this study. With the development of OA, the active ACSCs loss gradually as revealed by reduced BrdU label with time. But more considerable, the decrease in number of active ACSCs may be due to the increased inflammatory factors levels [1] and the cartilage destruction during OA development. This study showed ACSCs highly expressed positive MSC markers (CD44 and CD90), rarely expressed negative MSC markers (CD31, CD34, and CD45), and demonstrated high colony-forming efficiency. Analysis of the multipotent differentiation potential indicates that the osteogenesis and adipogenesis capabilities of isolated ACSCs were higher than that of the general chondrocytes, but weaker than that of the BM- MSCs. However, ACSCs exhibited higher chondrogenesis potential compared with BM-MSCs. Although BM-MSCs were thought to be a promising resource for cartilage regeneration, their use was associated with many serious problems, including low chondrogenesis potential, vascularization, and mineralization [32, 33]. A direct comparison study from Helen, and so forth, discovered that BMSCs resulted to a hypertrophic (endochondral) cartilage, and concluded that it can limit cartilage repair in case the tissue undergo mineralization [34]. In contrast to BM-MSCs, MSCs isolated from the auricular perichondrium displayed a highly chondrogenesis profile similar to that observed in chondrocytes [35]. Thus, the existence of ACSCs, especially because of their strong chondrogenesis tendency, provides a possibility for the use of intrinsic MSCs instead of exogenous cells in cartilage generation [21]; surgical transplant may be replaced in the future by ACSCs stimulation to regenerate the cartilage tissue. In healthy articular cartilage, the effects of TGFb1 are counterbalanced by the action of IL-1b, providing a balance in tissue homeostasis [36, 37]. During OA, high levels of IL-1b have been observed in synovial fluids and may actively participate in cartilage breakdown [38]. Bauge et al. expounded the mechanism of the IL-1b inhibition effect on the TGF-b/Smad pathway in chondrocytes [39]; however, little is known about the regulation of IL-1b on ACSCs chondrogenesis differentiation. In this study, we found that IL-1b suppressed TGF-b1 induced chondrogenesis in rat ACSCs in a dose-dependent manner. In addition, we investigated the mechanisms whereby IL-1b exerts its effects. The expression of several important factors was detected in the TGF-b/Smad pathway to obtain the key factors during the IL-1b-suppressed ACSCs chondrogenesis process. In this study, IL-1b can downregulate TGFbRII and upregulate Smad7 both at the mrna and protein levels in ACSCs. Given that the NF-jB pathway has a pivotal role in IL-1b responses, we investigated its role and its effects in ACSCs chondrogenesis. The involvement of the NF-jB pathway (particularly p65) was determined by its inhibitor BAY We found that IL-1b can activate the NF-jB pathway (particularly p65) efficiently, and the active p65 could suppress the ACSCs chondrogenesis process by TGF-bRII downregulation and Smad7 upregulation, we further confirmed that the IL-1b regulations of TGF-bRII and Smad7 were reversed by BAY treatment. This ordered process represents a potential molecular mechanism for the decline of cartilage self-regeneration caused by high levels of IL-1b and provided a possible therapeutic strategy for OA. The effect of IL-1b that impairs the responsiveness of ACSCs is dependent on the NF-jB pathway, as demonstrated through in vivo animal experiments. The percentage of activated ACSCs was significantly increased in the BAY treatment group compared with the OA control group. The active state maximized at approximately 30 days after the OAinducing surgical operation, in contrast to the control group, which maximized at about 14 days after OA induction. When the activated ACSCs of the control group became scarcely detectable on the 90th day, the percentage of activated ACSCs in the BAY treatment group was maintained at approximately 25%. The extent of cartilage degradation was significantly inhibited by BAY Although inhibiting NFjB pathway has previously been shown to antagonize some catabolic effects of IL-1b by suppressing NF-jB-regulated gene products involved in inflammation (cyclooxygenase-2, matrix metalloproteinase [MMP], vascular endothelial growth factor), inhibited apoptosis in chondrocyte [40]. However, whether inhibition of NF-jB can halt the progression of experimental OA in vivo has not been previously reported, this study leads to the following finding: IL-1b-induced suppression of ACSCs viability and proliferation is revoked by NF-jB inhibitor in vitro and in vivo, these effects may contribute to augment OA chondroprotection. It is absolutely possible that NF-jB inhibitor BAY mediates its effects by not only inhibiting catabolism but also increasing anabolism via targeting more than one cell signaling pathway in vivo, the detailed mechanism needs further single factor analysis in animal model. However, if this was the case, then the beneficial effects that inhibition of NF-jB might have in arthritis (OA and rheumatoid arthritis) therapy would be further emphasized as multitargeted therapy has a better chance of success against inflammation compared with therapies that aim for a single target [41]. CONCLUSIONS In summary, this study is the first to provide in vivo evidence of the existence of ACSCs and their action during early stage VC AlphaMed Press 2015

12 3136 In Vivo Identification and Induction of ACSCs of OA. IL-1b, which can be detected in the middle- and latestage OA, significantly impairs the responsiveness of ACSCs. These results presented a possible mechanism for both cartilage intrinsic repair and its final degradation. The NF-jB pathway contributes to this inhibition process and can rescue the ACSCs chondrogenesis, induce cartilage regeneration, and protect articular cartilage from injury in the OA animal model using a NF-jB pathway inhibitor. To the best of our knowledge, the NF-jB pathway inhibitor treatment used in OA is the first example to demonstrate the feasibility of inducing endogenous adult tissue-specific MSCs, ACSCs, for articular cartilage repair with far-reaching medical impact. ACKNOWLEDGMENTS This work was supported by grants from National Natural Science Foundation of China (No ), Chinese Academy of Sciences (No. XDA ), Science and Technology Commission of Shanghai Municipality (No and No ), Shanghai Municipal Commission of Health and Family Planning (No. 2013ZYJB0501), Shanghai Municipal Education Commission (Grant No.J50206), Shanghai Jiao Tong University (No.2013SMC-A-6), and National Institutes of Health (P20GM104937). AUTHOR CONTRIBUTIONS W.T.: conception and design, collection and assembly of data, data analysis and interpretation, and manuscript writing; Y.G.: conception and design and collection and assembly of data; X.Z.: conception and design, data analysis and interpretation, and manuscript writing; Y.H., Y.S., and S.X.: collection and assembly of data; N.Z.: data analysis and interpretation; Q.S., L.Q., Q.C., and K.D.: manuscript writing. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST The authors indicate no potential conflicts of interest. 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