Generation of Human-Induced Pluripotent Stem Cells From Anterior Cruciate Ligament.

Human-induced pluripotent stem cells (hiPSCs) are reprogrammed somatic cells and are an excellent cell source for tissue engineering applications, disease modeling, and for understanding human development. HiPSC lines have now been generated from a diverse range of somatic cell types and have been reported to retain an epigenetic memory of their somatic origin. To date, the reprogramming of a true ligament has not been reported. The aim of this study is to generate iPSCs from human anterior cruciate ligament (ACL) cells. ACL cells from three above-knee amputation donors, with donor matched dermal fibroblasts (DFs) were tested for reprogramming using an existing DF reprogramming protocol. ACL cells were, however, more sensitive than donor matched DF to transforming growth factor-ß (TGF-ß); displaying marked contraction, increased proliferation and increased TNC and COMP expression in vitro, which hindered reprogramming to iPSCs. Modification of the protocol by scoring the cell monolayer or by removal of TGF-ß during ACL reprogramming resulted in emerging colonies being easier to identify and extract, increasing reprogramming efficiency. Following 30 passages in culture, the generated ACL derived iPSCs displayed pluripotency markers, normal karyotype and can successfully differentiate to cells of the three embryonic germ layers. This study illustrates it is possible to generate hiPSCs from ligament and identifies optimized ligament reprogramming conditions. ACL derived iPSCs may provide a promising cell source for ligament and related tissue engineering applications. © 2019 The Authors. Journal of Orthopaedic Research® published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society J Orthop Res 38:92-104, 2020.

Stratification of knee osteoarthritis: two major patient subgroups identified by genome-wide expression analysis of articular cartilage.

INTRODUCTION: Osteoarthritis (OA) is a heterogeneous and complex disease. We have used a network biology approach based on genome-wide analysis of gene expression in OA knee cartilage to seek evidence for pathogenic mechanisms that may distinguish different patient subgroups. METHODS: Results from RNA-Sequencing (RNA-Seq) were collected from intact knee cartilage at total knee replacement from 44 patients with OA, from 16 additional patients with OA and 10 control patients with non-OA. Results were analysed to identify patient subsets and compare major active pathways. RESULTS: The RNA-Seq results showed 2692 differentially expressed genes between OA and non-OA. Analysis by unsupervised clustering identified two distinct OA groups: Group A with 24 patients (55%) and Group B with 18 patients (41%). A 10 gene subgroup classifier was validated by RT-qPCR in 16 further patients with OA. Pathway analysis showed increased protein expression in both groups. PhenomeExpress analysis revealed group differences in complement activation, innate immune responses and altered Wnt and TGFß signalling, but no activation of inflammatory cytokine expression. Both groups showed suppressed circadian regulators and whereas matrix changes in Group A were chondrogenic, in Group B they were non-chondrogenic with changes in mechanoreceptors, calcium signalling, ion channels and in cytoskeletal organisers. The gene expression changes predicted 478 potential biomarkers for detection in synovial fluid to distinguish patients from the two groups. CONCLUSIONS: Two subgroups of knee OA were identified by network analysis of RNA-Seq data with evidence for the presence of two major pathogenic pathways. This has potential importance as a new basis for the stratification of patients with OA for drug trials and for the development of new targeted treatments.

PhenomeScape: a cytoscape app to identify differentially regulated sub-networks using known disease associations.

PhenomeScape is a Cytoscape app which provides easy access to the PhenomeExpress algorithm to interpret gene expression data. PhenomeExpress integrates protein interaction networks with known phenotype to gene associations to find active sub-networks enriched in differentially expressed genes. It also incorporates cross-species phenotypes and associations to include results from animal models of disease. With expression data imported into PhenomeScape, the user can quickly generate and visualise interactive sub-networks. PhenomeScape thus enables researchers to use prior knowledge of a disease to identify differentially regulated sub-networks and to generate an overview of altered biologically processes specific to that disease. AVAILABILITY AND IMPLEMENTATION: Freely available for download at https://github.com/soulj/PhenomeScape CONTACT: jamie.soul@postgrad.manchester.ac.uk or jean-marc.schwartz@manchester.ac.uk.

Expression of Cannabinoid Receptors in Human Osteoarthritic Cartilage: Implications for Future Therapies.

Introduction: Cannabinoids have shown to reduce joint damage in animal models of arthritis and reduce matrix metalloproteinase expression in primary human osteoarthritic (OA) chondrocytes. The actions of cannabinoids are mediated by a number of receptors, including cannabinoid receptors 1 and 2 (CB1 and CB2), G-protein-coupled receptors 55 and 18 (GPR55 and GPR18), transient receptor potential vanilloid-1 (TRPV1), and peroxisome proliferator-activated receptors alpha and gamma (PPARα and PPARγ). However, to date very few studies have investigated the expression and localization of these receptors in human chondrocytes, and expression during degeneration, and thus their potential in clinical applications is unknown. Methods: Human articular cartilage from patients with symptomatic OA was graded histologically and the expression and localization of cannabinoid receptors within OA cartilage and underlying bone were determined immunohistochemically. Expression levels across regions of cartilage and changes with degeneration were investigated. Results: Expression of all the cannabinoid receptors investigated was observed with no change with grade of degeneration seen in the expression of CB1, CB2, GPR55, PPARα, and PPARγ. Conversely, the number of chondrocytes within the deep zone of cartilage displaying immunopositivity for GPR18 and TRPV1 was significantly decreased in degenerate cartilage. Receptor expression was higher in chondrocytes than in osteocytes in the underlying bone. Conclusions: Chondrocytes from OA joints were shown to express a wide range of cannabinoid receptors even in degenerate tissues, demonstrating that these cells could respond to cannabinoids. Cannabinoids designed to bind to receptors inhibiting the catabolic and pain pathways within the arthritic joint, while avoiding psychoactive effects, could provide potential arthritis therapies.

Cannabinoids: novel therapies for arthritis?

A key feature of osteoarthritis and rheumatoid arthritis is the loss of articular cartilage. Cartilage breakdown is mediated by complex interactions of proinflammatory cytokines, such as IL-1, inflammatory mediators, including nitric oxide and prostaglandin E(2), and proteases, including matrix metalloproteinases and aggrecanases, such as ADAMTS-4 and -5. Cannabinoids have been shown to reduce joint damage in animal models of arthritis. They have also been shown to prevent IL-1-induced matrix breakdown of collagen and proteoglycan, indicating that cannabinoids may mediate chondroprotective effects. Cannabinoids produce their effects via several cannabinoid receptors and it is important to identify the key cannabinoids and their receptors that are involved in chondroprotection. This review aims to outline the current and future prospects of cannabinoids as anti-arthritic therapeutics, in terms of their ability to prevent cartilage breakdown.