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Background Clinical translation of mesenchymal stromal cells (MSCs) necessitates basic characterization of the cell product since variability in biological source and processing of MSCs may impact therapeutic outcomes. Although expression of classical cell surface markers (e.g., CD90, CD73, CD105, and CD44) is used to define MSCs, identification of functionally relevant cell surface markers would provide more robust release criteria and options for quality control. In addition, cell surface expression may distinguish between MSCs from different sources, including bone marrow-derived MSCs and clinical-grade adipose-derived MSCs (AMSCs) grown in human platelet lysate (hPL). Methods In this work we utilized quantitative PCR, flow cytometry, and RNA-sequencing to characterize AMSCs grown in hPL and validated non-classical markers in 15 clinical-grade donors. Results We characterized the surface marker transcriptome of AMSCs, validated the expression of classical markers, and identified nine non-classical markers (i.e., CD36, CD163, CD271, CD200, CD273, CD274, CD146, CD248, and CD140B) that may potentially discriminate AMSCs from other cell types. More importantly, these markers exhibit variability in cell surface expression among different cell isolates from a diverse cohort of donors, including freshly prepared, previously frozen, or proliferative state AMSCs and may be informative when manufacturing cells. Conclusions Our study establishes that clinical-grade AMSCs expanded in hPL represent a homogeneous cell culture population according to classical markers,. Additionally, we validated new biomarkers for further AMSC characterization that may provide novel information guiding the development of new release criteria. Clinical trials Use of Autologous Bone Marrow Aspirate Concentrate in Painful Knee Osteoarthritis (BMAC): Clinicaltrials.gov NCT01931007 . Registered August 26, 2013. MSC for Occlusive Disease of the Kidney: Clinicaltrials.gov NCT01840540 . Registered April 23, 2013. Mesenchymal Stem Cell Therapy in Multiple System Atrophy: Clinicaltrials.gov NCT02315027 . Registered October 31, 2014. Efficacy and Safety of Adult Human Mesenchymal Stem Cells to Treat Steroid Refractory Acute Graft Versus Host Disease. Clinicaltrials.gov NCT00366145 . Registered August 17, 2006. A Dose-escalation Safety Trial for Intrathecal Autologous Mesenchymal Stem Cell Therapy in Amyotrophic Lateral Sclerosis. Clinicaltrials.gov NCT01609283 . Registered May 18, 2012.

Sampen HJI, Sharrocks AD, Lin X, et al. Open Access Rheumatol. 2009;1:151-161. We, the Editor and Publisher of the journal Open Access Rheumatology: Research and Reviews are issuing an expression of concern for the published article. Following publication, questions about the scientific integrity of the article were brought to the Publisher and Editors' attention. We reached out to the corresponding author who has been cooperative and as we continue to work through the issues raised, we advise readers to interpret the information presented in the article with due caution. The corresponding author has been notified about this Expression of Concern.

MicroRNAs (miRNAs) are critical post-transcriptional regulators of gene expression that control osteoblast mediated bone formation and osteoclast-related bone remodeling. Deregulation of miRNA mediated mechanisms is emerging as an important pathological factor in bone degeneration (eg, osteoporosis) and other bone-related diseases. MiRNAs are intriguing regulatory molecules that are networked with cell signaling pathways and intricate transcriptional programs through ingenuous circuits with remarkably simple logic. This overview examines key principles by which miRNAs control differentiation of osteoblasts as they evolve from mesenchymal stromal cells during osteogenesis, or of osteoclasts as they originate from monocytic precursors in the hematopoietic lineage during osteoclastogenesis. Of particular note are miRNAs that are temporally upregulated during osteoblastogenesis (eg, miR-218) or osteoclastogenesis (eg, miR-148a). Each miRNA stimulates differentiation by suppressing inhibitory signaling pathways (‘double-negative’ regulation). The excitement surrounding miRNAs in bone biology stems from the prominent effects that individual miRNAs can have on biological transitions during differentiation of skeletal cells and correlations of miRNA dysfunction with bone diseases. MiRNAs have significant clinical potential which is reflected by their versatility as disease-specific biomarkers and their promise as therapeutic agents to ameliorate or reverse bone tissue degeneration.

Although low-back pain is considered to be associated with cigarette smoking, the influence of cigarette smoking on the intervertebral discs (IVD) has not been confirmed. We established a rat model of passive cigarette smoking-induced IVD degeneration, and investigated the cytohistological changes in the IVD and the accompanying changes in gene expression. IVD from rats exposed to 8 weeks of passive cigarette smoking were stained with Elastica van Gieson, and exhibited marked destruction of the supportive structure of the reticular matrix in the nucleus pulposus (NP). Positive signals on safranin O, alcian blue, type II collagen and aggrecan staining were decreased in the destroyed structure. Safranin O and type II collagen signals were also decreased in the cartilage end-plate (CEP) after 4- and 8-weeks of cigarette smoking. In the CEP, the potential for apoptosis was increased significantly, as demonstrated by staining for single-strand DNA. However, there were no signs of apoptosis in the NP or annulus fibrosus cells. Based on these findings, we hypothesized that passive cigarette smoking-induced stress stimuli first affect the CEP through blood flow due to the histological proximity, thereby stimulating chondrocyte apoptosis and reduction of the extracellular matrix (ECM). This leads to reduction of the ECM in the NP, destroying the NP matrix, which can then progress to IVD degeneration.

Degradation of the extracellular matrix (ECM) by matrix metalloproteinases (MMPs) and release of basic fibroblast growth factor (bFGF) are principal aspects of the pathology of osteoarthritis (OA). ECM disruption leads to bFGF release, which activates the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) pathway and its downstream target the Ets-like transcription factor Elk-1. Previously we demonstrated that the bFGF-ERK-Elk-1 signaling axis is responsible for the potent induction of MMP-13 in human primary articular chondrocytes. Here we report that, in addition to phosphorylation of Elk-1, dynamic posttranslational modification of Elk-1 by small ubiquitin-related modifier (SUMO) serves as an important mechanism through which MMP-13 gene expression is regulated. We show that bFGF activates Elk-1 mainly through the ERK pathway and that increased phosphorylation of Elk-1 is accompanied by decreased conjugation of SUMO to Elk-1. Reporter gene assays reveal that phosphorylation renders Elk-1 competent for induction of MMP-13 gene transcription, while sumoylation has the opposite effect. Furthermore, we demonstrate that the SUMO-conjugase Ubc9 acts as a key mediator for Elk-1 sumoylation. Taken together, our results suggest that sumoylation antagonizes the phosphorylation-dependent transactivation capacity of Elk-1. This attenuates transcription of its downstream target gene MMP-13 to maintain the integrity of cartilage ECM homeostasis.

Background: Chronic allograft rejection is the major problem encountered in organ transplantation and is the end point of several complex processes, accounting for almost 30% of the renal allograft recipients returning for a second allograft. HLA antibodies play a major role in the recipient's immune response against the renal allograft. However, importance of HLA antibodies in transplantation is well documented, but despite the very sensitive HLA screening assays, antibody-mediated allograft rejection still occurs without detectable HLA antibodies. Numerous non-HLA antibodies have been identified in renal organ transplantation, Vimentin is an ideal candidate non-HLA antigen for antibody-mediated rejection. We focused on antibodies against vimentin and its isoforms which could alter the cytoskeletal structural integrity, which in turn could lead to the development of irreversible fibrosis in the allograft. Method: Generation of vimentin fragments were achieved via chemical treatment (Cyanogen Bromide, Endoproteinase lys-c). Biological isoforms were obtained from lymphocytes treated MHC Class II antibody (directed against HLA-DR) and anti-CD3. Dot- blot and western blot assays with chemilumniscence detection methods were used to identify the protein and fragments. Immunological reactivity of fragments generated by via chemical treatment and biological isoforms were determined by using polyclonal anti-human vimentin antibody. A cohort was comprised of 33 patients' serum tested in this study; including chronic rejection (n=6), Polyomavirus nephropathy (n=5), and T-cell mediated rejection (n=9), antibody mediated rejection C4d (n=5) positive and C4d negative (n=4). All these were diagnosed by histopathology and Immunohistochemistry of allograft biopsies read by a qualified board certified pathologist at Rush University Medical Center. Western blot assays with chemilumniscence detection methods were used to determine the immunological reactivity of pooled patients' sera from various types of allograft rejection to the vimentin pooled fraction. Luminex based assays were developed to detect antibodies against different pooled fraction of pure vimentin generated by cyanogen bromide digestion. With Luminex single fragment bead technology, patients-serum antibodies against vimentin and isoform could be identified before a rise in serum creatinine and graft failure. Results: Western blot assays in both biologically induced isoforms and chemical induced fractions showed several bands of varying intensities in biologically induced isoforms and in vitro Generated fragments; the predominant form in both biological and in vitro generated fragments was the one around. 37kDa. Also, there was an approximately 17kDa fragment that was seen in the HLA-DR antibody stimulated lymphocytes. An approximately 50kDa and 37kDa bands showed with varying in the intensities in different groups of chronic rejection patients. Preliminary evaluation of the assays was developed to pooled fractions for vimentin autoantibodies. Conclusion: A direct capture assay for vimentin pooled fractions autoantibodies was developed and analytically validated. One-way a nova analysis was showing the significant differences in autoantibody levels between groups P value <0.05. Preliminary evaluation of those assays against of 33 allograft patient's serum was promising and justifies additional testing with larger cohorts in future studies.

Osteoprotegerin is basic glycoprotein that in humans is encoded by the TNFRSF 11 B gene. It is produced by osteoblasts, chondrocytes and other mesenchymal cells, lacks a transmembrane domain and acts as a secreted decoy receptor which is a member of the tumor necrosis factor (TNF) receptor superfamily that has no direct signaling capacity. OPG acts by binding to its natural ligand OPGL, which is also known as RANKL (receptor activator of NF-kappa B ligand). This binding prevents RANKL from activating its cognate receptor RANK, which is the osteoclast receptor vital for osteoclast differentiation, activation and survival. Synthesis of cartilage by chondrocytes is one of the important steps for endochondral bone formation. Osteoprotegerin is critical for bone development and remodeling and has been shown to be required for osteoclast function. However, the role that chondrocyte OPG plays in cartilage and skeletal development as well as its underlying cellular and molecular mechanisms are not well understood. By using in vivo and in vitro strategies we expect that OPG is a physiologically important regulator in chondrocytes differentiation, modeling and remodeling through its effect on chondroclasts and or osteoclasts. This hypothesis is based on the expression of RANKL by chondrocytes which may support chondroclasts differentiation by a mechanism resembling osteoblastdependent osteoclastogenesis but using a different molecular processes requiring Osteoprotegerin for endochondral ossification.

Biglycan is a small, leucine-rich repeat proteoglycan that has been shown to be elevated in the intervertebral discs (IVDs) of older adults (Cs-Szabo, 2002) and can interact with many different signaling molecules and structural proteins. TNF-alpha (TNF) is a general modulator of inflammation and may be a trigger in the chronic inflammatory response that leads to disc degeneration. Since biglycan has been shown to interact with TNFalpha (Tufvesson and Westergren-Thorsson, 2002), it may play a role in modulating its response in the IVD. Western (NFkB) and RT-PCR (MMP-1, -13, NGF and VEGF) assays were performed on both human and bovine cultured cells from IVD tissue. Treatment with biglycan alone was found to be almost indistinguishable from control groups; while biglycan in combination with TNF-alpha was able to significantly reduce transcription and expression response when compared with just TNF-alpha. Biglycan may help modulate the effects of TNF-alpha in the IVD.

Recurrent hemarthrosis resulting in arthropathy remains the most common and debilitating complication of hemophilia. This is true despite prophylactic factor administration and especially in patients with inhibitors. The pathobiological mechanisms remain unclear. Neutrophils are the most abundant leukocytes and are the first cells to arrive at the site of infection or inflammation. The main physiological and pathogenic activities of neutrophils include adherence and migration, degranulation and release of inflammatory mediators, phagocytosis and apoptosis. From our previous histological analysis in the murine model of joint bleeding, we found a high rate of infiltration of neutrophils in the synovial space. Since the role of neutrophils is not clear in the development of the proliferative synovitis that develops after hemarthrosis, this study aimed to address this gap in knowledge by studying the effects of neutrophil depletion by injecting anti-Ly6G (1A8) antibody on the development of synovitis in mice deficient in factor 8 gene expression compared to isotype IgG, which will be used as a control. Neutrophil depleted (1A8 treated) and control (IgG treated) animals were subjected to a single massive experimental joint hemorrhage and then sacrificed 6 hours after induction of Hemarthrosis and on day 1, 2, 3 or 7 after the hemarthrosis and assessed for the development of synovitis. Knee joint diameter was measured as a reflection of inflammation. The joints were then isolated and subjected to histological analysis and enumeration of the neutrophil density in the synovium of lA8 and control antibody-treated mice. The degree of neutrophil infiltration was prominent on days 13, and decreased on day 7 and 14 when the joint disease was established. Though the degree of inflammation is reduced in the initial days, the level of joint disease in the two groups is comparable by Day 14. This could be due to macrophages that infiltrate the joint space as seen in the histological sections that could compensate for the pro-inflammatory cytokines leading to the same level of joint disease in both groups. Although the involvement of macrophages in the development of arthritis was not examined, macrophages might play some role in the pathogenesis of this arthritis model.

ADAMTS5 (TS5), as a member of the ADAMTS-aggrecanase family of proteases is considered to be largely responsible for the turnover of aggrecan and versican in vivo. Its ablation has been shown to protect murine cartilage from a variety of joint injury models, give rise to heart valve abnormalities and cause defective wound healing, as well as affect fibroblast-myofibroblast transition due to altered versican turnover. However, a potentially broader cell biological role for this protein in signal transduction to regulate extracellular matrix turnover during tissue homeostasis and regeneration are not well understood. We have previously reported that ADAMTS5 KO mice exhibit cartilage protection after joint injury due to reduced fibrosis of the joint capsule and synovium and enhanced articular cartilage deposition. Additionally, these mice display impaired wound healing of the skin and tendons, and this is associated with aggrecan-rich deposits at the wound sites in vivo and altered TGFb signaling in fibroblasts in vitro. We hypothesize that the multi-tissue chondrogenic wound-healing response in ADAMTS5 KO mice is due to a nodal role of TS5 in regulating the differentiation of progenitor cells into fibroblastic cell types. To address this hypothesis, we have established monolayer cultures of adipose derived stromal cells (ADSCs) from wild type and two strains of ADAMTS5 KO mice and have shown their basal capacity in vitro for versican, aggrecan, hyaluronan and collagen type I synthesis. Aggrecan synthesis was stimulated by increased (10 mM) extracellular glucose, and this was more pronounced in TS5 KO cells, which exhibited enhanced glucose uptake, under both physiological (5mM) and hyperglycemic (10mM) conditions. We also investigated the catabolic turnover of aggrecan and versican in the stromal cell cultures. Both were degraded by ADAMTS-aggrecanases, and no qualitative or quantitative differences in their catabolism were detected between WT and TS5 KO cells. Notably, ADAMTS-cleavage of the core proteins occurred largely intra-cellularly during the secretory process, and took place both in the absence and presence of fetal bovine serum. Furthermore, a comparative analysis of aggrecan turnover in epiphyseal chondrocytes showed no differences in catabolic activity against the proteoglycan in WT and TS5 KO cells, we propose that ADAMTS5 is not essential for aggrecan cleavage in these two cells types. In keeping with previous work on newborn skin fibroblasts we found that stromal cells from ADAMTS5 KO mice exhibited altered TOFb1 responsiveness, which in this study was shown by lack of TGFb1 stimulated hyaluronan synthase 1 and 2 gene expression and collagen type I synthesis, in TS5 KO cultures compared to WT. A non-proteolytic function for the TS5 protein was further underscored by our finding, that only fragments of TS5 were detectable in the cell associated matrix of WT stromal cells. Their absence in TS5 KO cells, their lack of proteolytic activity (size of 30kDa and 40 kDa, by reactivity on western blot analyses with 2 peptide specific antibodies), and their altered abundance in cells treated with the endocytotic/endosomal trafficking inhibitors, Dynasore or Bafilomycin, indicates a novel cell-biological role for TS5 in modulating signal transduction mechanisms by regulating growth factor receptor endocytosis and/or endosomal recycling.