Interleukin‐1β induces differentiation of human mesenchymal stem cells into osteoblasts via the Wnt‐5a/receptor tyrosine kinase–like orphan receptor 2 pathway

Objective

Mesenchymal stem cells (MSCs) are considered to be a novel tool for the treatment of rheumatoid arthritis (RA) because of their multipotency to differentiate into osteoblasts and chondrocytes, their immunosuppressive effects, and availability. The aim of this study was to assess the mechanisms of human MSC differentiation into osteoblasts under inflammatory conditions.

Methods

Human MSCs were cultured in commercialized osteogenic induction medium with inflammatory cytokines for up to 10 days. Osteoblast differentiation was detected by alkaline phosphatase staining and messenger RNA (mRNA) expression of multiple osteoblast markers. Mineralization was assessed by alizarin red S staining.

Results

Among the various cytokines tested, interleukin‐1β (IL‐1β) induced differentiation of human MSCs into osteoblasts, which was confirmed by alkaline phosphatase activity, expression of RUNX2 mRNA, and strong alizarin red S staining. Among various molecules of the Wnt family, Wnt‐5a and receptor tyrosine kinase–like orphan receptor 2 (Ror2), a major receptor of Wnt‐5a, were significantly induced in human MSCs by IL‐1β. Silencing of either WNT5A or ROR2 by small interfering RNA with 2 different sequences reduced alkaline phosphatase activity, RUNX2 expression, and alizarin red S staining of human MSCs induced by IL‐1β.

Conclusion

IL‐1β effectively and rapidly induced human MSC differentiation into osteoblasts and mineralization, mainly through the noncanonical Wnt‐5a/Ror2 pathway. These results suggest potential benefits of IL‐1β–treated human MSCs in the treatment of damaged bone as well as in the induction of self‐renewal and self‐repair of damaged tissue, including osseous tissue.

     

In vitro stage‐specific chondrogenesis of mesenchymal stem cells committed to chondrocytes

Objective

Osteoarthritis is characterized by an imbalance in cartilage homeostasis, which could potentially be corrected by mesenchymal stem cell (MSC)–based therapies. However, in vivo implantation of undifferentiated MSCs has led to unexpected results. This study was undertaken to establish a model for preconditioning of MSCs toward chondrogenesis as a more effective clinical tool for cartilage regeneration.

Methods

A coculture preconditioning system was used to improve the chondrogenic potential of human MSCs and to study the detailed stages of chondrogenesis of MSCs, using a human MSC line, Kp‐hMSC, in commitment cocultures with a human chondrocyte line, hPi (labeled with green fluorescent protein [GFP]). In addition, committed MSCs were seeded into a collagen scaffold and analyzed for their neocartilage‐forming ability.

Results

Coculture of hPi‐GFP chondrocytes with Kp‐hMSCs induced chondrogenesis, as indicated by the increased expression of chondrogenic genes and accumulation of chondrogenic matrix, but with no effect on osteogenic markers. The chondrogenic process of committed MSCs was initiated with highly activated chondrogenic adhesion molecules and stimulated cartilage developmental growth factors, including members of the transforming growth factor β superfamily and their downstream regulators, the Smads, as well as endothelial growth factor, fibroblast growth factor, insulin‐like growth factor, and vascular endothelial growth factor. Furthermore, committed Kp‐hMSCs acquired neocartilage‐forming potential within the collagen scaffold.

Conclusion

These findings help define the molecular markers of chondrogenesis and more accurately delineate the stages of chondrogenesis during chondrocytic differentiation of human MSCs. The results indicate that human MSCs committed to the chondroprogenitor stage of chondrocytic differentiation undergo detailed chondrogenic changes. This model of in vitro chondrogenesis of human MSCs represents an advance in cell‐based transplantation for future clinical use.

     

Large‐scale extraction and characterization of CD271+ multipotential stromal cells from trabecular bone in health and osteoarthritis: Implications for bone regeneration strategies based on uncultured or minimally cultured multipotential stromal cells

Objective

To test the hypothesis that CD45^lowCD271+ bone marrow multipotential stromal cells (MSCs) are abundant in the trabecular bone niche and to explore their functional “fitness” in health and osteoarthritis (OA).

Methods

Following enzymatic extraction, MSC release was evaluated using colony‐forming unit–fibroblast (CFU‐F) and colony‐forming unit–osteoblast assays, flow cytometry, and confocal microscopy. CD45^lowCD271+ cells isolated by fluorescence‐activated cell sorting were enumerated and expanded under standard and clonal conditions. Their proliferative and osteogenic potencies were assessed in relation to donor age and compared with those of aspirated CD45^lowCD271+ cells. In vitro and in vivo MSC “aging” was measured using quantitative polymerase chain reaction–based telomere length analysis, and standard differentiation assays were utilized to demonstrate multipotentiality.

Results

Cellular isolates from trabecular bone cavities contained ∼65‐fold more CD45^lowCD271+ cells compared with aspirates (P < 0.0001) (median 1.89% [n = 39] and 0.029% [n = 46], respectively), concordant with increased CFU‐F release. Aspirated and enzymatically released CD45^lowCD271+ cells had identical MSC phenotypes (∼100% CD73+CD105+CD13+, ∼50–60% CD146+CD106+CD166+) and contained large proportions of highly clonogenic multipotential cells. In vitro osteogenic potency of freshly isolated CD45^lowCD271+ cells was comparable with, and often above, that of early‐passage MSCs (8–14%). Their frequency and in vivo telomere status in OA bone were similar to those in bone from age‐matched controls.

Conclusion

Our findings show that CD45^lowCD271+ MSCs are abundant in the trabecular bone cavity and indistinguishable from aspirated CD45^lowCD271+ MSCs. In OA they display aging‐related loss of proliferation but no gross osteogenic abnormality. These findings offer new opportunities for direct study of MSCs in musculoskeletal diseases without the requirement for culture expansion. They are also relevant for direct therapeutic exploitation of prospectively isolated, minimally cultured MSCs in trauma and OA.

     

Adoptive Transfer of Human Gingiva‐Derived Mesenchymal Stem Cells Ameliorates Collagen‐Induced Arthritis via Suppression of Th1 and Th17 Cells and Enhancement of Regulatory T Cell Differentiation

Objective

Current approaches offer no cures for rheumatoid arthritis (RA). Accumulating evidence has revealed that manipulation of bone marrow–derived mesenchymal stem cells (BM‐MSCs) may have the potential to control or even prevent RA, but BM‐MSC–based therapy faces many challenges, such as limited cell availability and reduced clinical feasibility. This study in mice with established collagen‐induced arthritis (CIA) was undertaken to determine whether substitution of human gingiva‐derived mesenchymal stem cells (G‐MSCs) would significantly improve the therapeutic effects.

Methods

CIA was induced in DBA/1J mice by immunization with type II collagen and Freund's complete adjuvant. G‐MSCs were injected intravenously into the mice on day 14 after immunization. In some experiments, intraperitoneal injection of PC61 (anti‐CD25 antibody) was used to deplete Treg cells in arthritic mice.

Results

Infusion of G‐MSCs in DBA/1J mice with CIA significantly reduced the severity of arthritis, decreased the histopathology scores, and down‐regulated the production of inflammatory cytokines (interferon‐γ and interleukin‐17A). Infusion of G‐MSCs also resulted in increased levels of CD4+CD39+FoxP3+ cells in arthritic mice. These increases were noted early after infusion in the spleens and lymph nodes, and later after infusion in the synovial fluid. The FoxP3+ Treg cells that were increased in frequency mainly consisted of Helios‐negative cells. When Treg cells were depleted, infusion of G‐MSCs partially interfered with the progression of CIA. Pretreatment of G‐MSCs with a CD39 or CD73 inhibitor significantly reversed the protective effect of G‐MSCs on CIA.

Conclusion

The role of G‐MSCs in controlling the development and severity of CIA mostly depends on CD39/CD73 signals and partially depends on the induction of CD4+CD39+FoxP3+ Treg cells. G‐MSCs provide a promising approach for the treatment of autoimmune diseases.

     

Reversal of serologic,immunologic, and histologic dysfunction in mice with systemic lupus erythematosus by long‐term serial adipose tissue–derived mesenchymal stem cell transplantation

Objective

To investigate the efficacy of human adipose tissue–derived mesenchymal stem cell (AD‐MSC) transplantation in systemic lupus erythematosus (SLE) and to determine the optimal transplantation window for stem cells either before or after disease onset.

Methods

(NZB × NZW)F₁ mice with SLE were administered human AD‐MSCs (5 × 10⁵) intravenously every 2 weeks from age 6 weeks until age 60 weeks, while the control group received saline vehicle on the same schedule. Another experiment was carried out with a different initiation time point for serial transplantation (age 6 weeks or age 32 weeks).

Results

Long‐term serial administration (total of 28 times) of human AD‐MSCs ameliorated SLE without any adverse effects. Compared with the control group, the human AD‐MSC–treated group had a significantly higher survival rate with improvement of histologic and serologic abnormalities and immunologic function, and also had a decreased incidence of proteinuria. Anti–double‐stranded DNA antibodies and blood urea nitrogen levels decreased significantly with transplantation of human AD‐MSCs, and serum levels of granulocyte–macrophage colony‐stimulating factor, interleukin‐4 (IL‐4), and IL‐10 increased significantly. A significant increase in the proportion of CD4+FoxP3+ cells and a marked restoration of capacity for cytokine production were observed in spleens from the human AD‐MSC–treated group. In the second experiment, an early stage treatment group showed better results (higher survival rates and lower incidence of proteinuria) than an advanced stage treatment group.

Conclusion

Serial human AD‐MSC transplantation had beneficial effects in the treatment of SLE, without adverse effects. Transplantation of human AD‐MSCs before disease onset was preferable for amelioration of SLE and restoration of immune homeostasis.

     

Cell therapy using allogeneic bone marrow mesenchymal stem cells prevents tissue damage in collagen‐induced arthritis

Objective

Mesenchymal stem cells (MSCs) are precursors of tissue of mesenchymal origin, but they also have the capacity to regulate the immune response by suppressing T and B lymphocyte proliferation in a non–major histocompatibility complex–restricted manner. Use of MSCs as immunosuppressant agents in autoimmune diseases has been proposed and successfully tested in animal models. We explored the feasibility of using allogeneic MSCs as therapy for collagen‐induced arthritis, a mouse model for human rheumatoid arthritis.

Methods

DBA/1 mice were immunized with type II collagen in Freund's complete adjuvant, and some of the animals received an intraperitoneal injection of allogeneic MSCs.

Results

A single injection of MSCs prevented the occurrence of severe, irreversible damage to bone and cartilage. MSCs induced hyporesponsiveness of T lymphocytes as evidenced by a reduction in active proliferation, and modulated the expression of inflammatory cytokines. In particular, the serum concentration of tumor necrosis factor α was significantly decreased. MSCs exerted their immunomodulatory function by educating antigen‐specific Tregs.

Conclusion

Our results suggest an effective new therapeutic approach to target the pathogenic mechanism of autoimmune arthritis using allogeneic MSCs. However, further studies are required before these results can be translated to clinical settings.

     

Inhibition of interleukin‐6 receptor directly blocks osteoclast formation in vitro and in vivo

Objective

To investigate the efficacy of a murine anti–interleukin‐6 receptor (anti–IL‐6R) antibody in directly blocking tumor necrosis factor (TNF)– and RANKL‐mediated osteoclastogenesis in vitro and in vivo.

Methods

The efficacy of a murine antibody against IL‐6R in blocking osteoclast differentiation of mononuclear cells stimulated with RANKL was tested. In addition, arthritic human TNFα–transgenic mice were treated with anti–IL‐6R antibody, and osteoclast formation and bone erosion were assessed in arthritic paws.

Results

Blockade of IL‐6R dose dependently reduced osteoclast differentiation and bone resorption in monocyte cultures stimulated with RANKL or RANKL plus TNF. In human TNFα–transgenic mice, IL‐6R blockade did not inhibit joint inflammation, but it strongly reduced osteoclast formation in inflamed joints as well as bone erosion in vivo. Neither the cell influx into joints nor the synovial expression of IL‐6 and RANKL changed with IL‐6R blockade, while the synovial expression of IL‐1 was significantly reduced. In contrast, TNF‐mediated systemic bone loss was not inhibited by IL‐6R blockade.

Conclusion

These data suggest that blockade of IL‐6R directly affects osteoclast formation in vitro and in vivo, suggesting a direct and specific effect of anti–IL‐6R therapy on osteoclasts independently of its antiinflammatory effects. This effect adds significantly to the structure‐sparing potential of pharmacologic blockade of IL‐6R in arthritis.

     

Localization of 3‐nitrotyrosine to rheumatoid and normal synovium

Objective

To determine the localization of 3‐nitrotyrosine (3‐NT), a footprint marker of peroxynitrite (ONOO⁻) and other reactive nitrogen species, to the inflamed human synovium and to compare this with normal synovial and nonsynovial tissue of human and animal origin.

Methods

Monoclonal and polyclonal antibodies were used to investigate for 3‐NT, inducible nitric oxide synthase (iNOS), macrophage marker CD68, and the vascular smooth muscle marker α‐actin by avidin–biotin immunocytochemistry.

Results

In the inflamed synovium, 3‐NT was found in the vascular smooth muscle and macrophages. In normal human synovium, 3‐NT was present in the vascular smooth muscle and some lining cells and was not associated with immunoreactivity for iNOS. Similarly, 3‐NT could be demonstrated in the vascular smooth muscle cells of normal rats and iNOS knockout mice. It was not present in the vascular smooth muscle of healthy, nonsynovial tissue.

Conclusion

The synovial vasculature in histologically normal human and naive rodent synovium was alone among the normal tissues studied in exhibiting iNOS‐independent immunoreactivity for 3‐NT. These findings suggest a physiologic role for ONOO⁻ in normal synovial vascular function.

     

Osteoarthritic chondrocyte–secreted morphogens induce chondrogenic differentiation of human mesenchymal stem cells

Objective

The potential of stem cells to repair compromised cartilage tissue, such as in osteoarthritis (OA), depends strongly on how transplanted cells respond to factors secreted from the residing OA chondrocytes. This study was undertaken to determine the effect of morphogenetic signals from OA chondrocytes on chondrogenic differentiation of human mesenchymal stem cells (MSCs).

Methods

The effect of OA chondrocyte–secreted morphogens on chondrogenic differentiation of human MSCs was evaluated using a coculture system involving both primary and passaged OA chondrocytes. The findings were compared against findings for human MSCs cultured in OA chondrocyte–conditioned medium. Gene expression analysis, biochemical assays, and immunofluorescence staining were used to characterize the chondrogenic differentiation of human MSCs. Mass spectrometry analysis was used to identify the soluble factors. Numerical analysis was carried out to model the concentration profile of soluble factors within the human MSC–laden hydrogels.

Results

The human MSCs cocultured with primary OA chondrocytes underwent chondrogenic differentiation even in the absence of growth factors; however, the same effect could not be mimicked using OA chondrocyte–conditioned medium or expanded cells. Additionally, the cocultured environment down‐regulated hypertrophic differentiation of human MSCs. Mass spectrometry analysis demonstrated cell–cell communication and chondrocyte phenotype–dependent effects on cell‐secreted morphogens.

Conclusion

The experimental findings, along with the results of the numerical analysis, suggest a crucial role of soluble morphogens and their local concentrations in the differentiation pattern of human MSCs in a 3‐dimensional environment. The concept of using a small number of chondrocytes to promote chondrogenic differentiation of human MSCs while preventing their hypertrophic differentiation could be of great importance in formulating effective stem cell–based cartilage repair.

     

Suppression of leukocyte infiltration and cartilage degradation by selective inhibition of pre–B cell colony‐enhancing factor/visfatin/nicotinamide phosphoribosyltransferase: Apo866‐mediated therapy in human fibroblasts and murine collagen‐induced arthritis

Objective

To assess the ability of pre–B cell colony‐enhancing factor (PBEF) to regulate inflammation and degradative processes in inflammatory arthritis, using the small molecule inhibitor APO866 in human fibroblasts in vitro and in murine collagen‐induced arthritis (CIA).

Methods

Enzyme‐linked immunosorbent assays were used to examine regulation of expression of metalloproteinases and chemokines in human fibroblasts. The role of PBEF was further examined using APO866 in mice with CIA, with effects on disease activity assessed using radiography, histology, in vivo imaging, and quantitative polymerase chain reaction (qPCR).

Results

In vitro activation of human fibroblasts with PBEF promoted expression of matrix metalloproteinase 3 (MMP‐3), CCL2, and CXCL8, an effect inhibited by APO866. In mice with CIA, early intervention with APO866 inhibited synovial inflammation, including chemokine‐directed leukocyte infiltration, and reduced a systemic marker of inflammation, serum hyaluronic acid. APO866 blockade led to reduced expression of MMP‐3 and MMP‐13 in joint extracts and to a reduction in a systemic marker of cartilage erosion, serum cartilage oligomeric matrix protein. Radiologic images revealed that APO866 protected against bone erosion, while qPCR demonstrated inhibition of RANKL expression. In mice with established disease, APO866 reduced synovial inflammation and cartilage destruction, and halted bone erosion. In addition, APO866 reduced the activity of MMP‐3, CCL2, and RANKL in vivo, and inhibited production of CCL2 and RANKL in synovial explants from arthritic mice, a result that was reversed with nicotinamide mononucleotide.

Conclusion

These findings confirm PBEF to be an important regulator of inflammation, cartilage catabolism, and bone erosion, and highlight APO866 as a promising therapeutic agent for targeting PBEF activity in inflammatory arthritis.

     

Identification of synovium‐specific homing peptides by in vivo phage display selection

Objective

To identify homing peptides specific for human synovium that could be used as targeting devices for delivering therapeutic/diagnostic agents to human joints.

Methods

Human synovium and skin were transplanted into SCID mice. A disulfide‐constrained 7–amino acid peptide phage display library was injected intravenously into the animals and synovial homing phage recovered from synovial grafts. Following 3–4 cycles of enrichment, DNA sequencing of homing phage clones allowed the identification of specific peptides that were synthesized by a‐fluorenylmethyloxycarbonyl chemistry and used in competitive in vivo assays and immunohistochemistry analyses.

Results

We isolated synovial homing phages displaying specific peptides that distinctively bound to synovial but not skin or mouse microvascular endothelium (MVE). They retained their tissue homing specificity in vivo, independently from the phage component, the original pathology of the transplanted tissue, and the degree of human/murine graft vascularization. One such peptide (CKSTHDRLC) maintained synovial homing specificity both when presented by the phage and as a free synthetic peptide. The synthetic peptide also competed with and inhibited in vivo the binding of the parent phage to the cognate synovial MVE ligand.

Conclusion

This is the first report describing peptides with homing properties specific for human synovial MVE. This was demonstrated using a novel approach targeting human tissues, transplanted into SCID mice, directly by in vivo phage display selection. The identification of such peptides opens the possibility of using these sequences to construct joint‐specific drug delivery systems that may have considerable impact in the treatment of arthritic conditions.

     

MicroRNA‐140 is expressed in differentiated human articular chondrocytes and modulates interleukin‐1 responses

Objective

MicroRNA (miRNA) are a class of noncoding small RNAs that act as negative regulators of gene expression. MiRNA exhibit tissue‐specific expression patterns, and changes in their expression may contribute to pathogenesis. The objectives of this study were to identify miRNA expressed in articular chondrocytes, to determine changes in osteoarthritic (OA) cartilage, and to address the function of miRNA‐140 (miR‐140).

Methods

To identify miRNA specifically expressed in chondrocytes, we performed gene expression profiling using miRNA microarrays and quantitative polymerase chain reaction with human articular chondrocytes compared with human mesenchymal stem cells (MSCs). The expression pattern of miR‐140 was monitored during chondrogenic differentiation of human MSCs in pellet cultures and in human articular cartilage from normal and OA knee joints. We tested the effects of interleukin‐1β (IL‐1β) on miR‐140 expression. Double‐stranded miR‐140 (ds–miR‐140) was transfected into chondrocytes to analyze changes in the expression of genes associated with OA.

Results

Microarray analysis showed that miR‐140 had the largest difference in expression between chondrocytes and MSCs. During chondrogenesis, miR‐140 expression in MSC cultures increased in parallel with the expression of SOX9 and COL2A1. Normal human articular cartilage expressed miR‐140, and this expression was significantly reduced in OA tissue. In vitro treatment of chondrocytes with IL‐1β suppressed miR‐140 expression. Transfection of chondrocytes with ds–miR‐140 down‐regulated IL‐1β–induced ADAMTS5 expression and rescued the IL‐1β–dependent repression of AGGRECAN gene expression.

Conclusion

This study shows that miR‐140 has a chondrocyte differentiation–related expression pattern. The reduction in miR‐140 expression in OA cartilage and in response to IL‐1β may contribute to the abnormal gene expression pattern characteristic of OA.

     

Regulation of angiogenesis by the C‐X‐C chemokines interleukin‐8 and epithelial neutrophil activating peptide 78 in the rheumatoid joint

Objective

Angiogenesis, the growth of new blood vessels, is vital to the ingress of inflammatory leukocytes in rheumatoid arthritis (RA) synovial tissue and to the growth and proliferation of RA pannus. The factors that mediate the growth of new blood vessels have not been completely defined. This study examined the ability of Glu‐Leu‐Arg (ELR)–containing chemokines to induce angiogenesis in the RA joint.

Methods

To reflect angiogenic activity in vivo, we selected a model using whole human synovial tissue rather than isolated cells. Tissues were examined by immunohistochemistry and enzyme‐linked immunosorbent assay, and tissue homogenates were immunoneutralized and assayed for their ability to induce endothelial cell chemotaxis and rat corneal neovascularization.

Results

Cells expressing interleukin‐8 (IL‐8) and epithelial neutrophil activating peptide 78 (ENA‐78) were located in proximity to factor VIII–related antigen–immunopositive endothelial cells. RA homogenates produced more IL‐8 and ENA‐78 compared with normal synovial tissue homogenates. Moreover, homogenates from RA synovial tissue produced significantly more chemotactic activity for endothelial cells in vitro and angiogenic activity in the rat cornea in vivo than did normal synovial tissue homogenates. The effects of IL‐8 and ENA‐78 accounted for a significant proportion of the chemotactic activity of endothelial cells and angiogenic activity found in RA synovial tissue homogenates.

Conclusion

These results indicate that the ELR‐containing chemokines IL‐8 and ENA‐78 are important contributors to the angiogenic activity found in the inflamed RA joint. It is possible that efforts aimed at down‐regulating these chemokines offer a novel targeted therapy for the treatment of RA.

     

Expression and proinflammatory role of proteinase‐activated receptor 2 in rheumatoid synovium: Ex vivo studies using a novel proteinase‐activated receptor 2 antagonist

Objective

Serine proteinases activate the G protein–coupled receptor, proteinase‐activated receptor 2 (PAR‐2), via cleavage and exposure of a tethered ligand. PAR‐2 is known to exert proinflammatory actions in a murine model of arthritis, since PAR‐2–deficient mice exhibit strikingly reduced articular inflammation. This study was undertaken to examine synovial PAR‐2 expression and to determine the effect of a novel PAR‐2 antagonist on synovial cytokine production, in order to investigate the hypothesis that PAR‐2 plays a critical role in the pathogenesis of rheumatoid arthritis (RA).

Methods

Using a monoclonal antibody to human PAR‐2, expression in RA synovium and cultured synovial fibroblasts was characterized. The novel PAR‐2 antagonist, ENMD‐1068, was added to primary cultures of RA synovial tissue, from which spontaneous cytokine release was measured.

Results

PAR‐2 was substantially up‐regulated in RA synovium compared with control synovial tissue from patients with osteoarthritis or seronegative inflammatory arthritis, neither of which exhibited significant PAR‐2 expression. Importantly, spontaneous release of tumor necrosis factor α and interleukin‐1β from RA synovium was substantially inhibited by ENMD‐1068, in a dose‐dependent manner.

Conclusion

These findings identify PAR‐2 as a novel upstream regulator of proinflammatory cytokine production in RA and indicate its potential as a novel therapeutic target in inflammatory arthritis.