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.

SkeletalVis: an exploration and meta-analysis data portal of cross-species skeletal transcriptomics data.

MOTIVATION: Skeletal diseases are prevalent in society, but improved molecular understanding is required to formulate new therapeutic strategies. Large and increasing quantities of available skeletal transcriptomics experiments give the potential for mechanistic insight of both fundamental skeletal biology and skeletal disease. However, no current repository provides access to processed, readily interpretable analysis of this data. To address this, we have developed SkeletalVis, an exploration portal for skeletal gene expression experiments. RESULTS: The SkeletalVis data portal provides an exploration and comparison platform for analysed skeletal transcriptomics data. It currently hosts 287 analysed experiments with 739 perturbation responses with comprehensive downstream analysis. We demonstrate its utility in identifying both known and novel relationships between skeletal expression signatures. SkeletalVis provides users with a platform to explore the wealth of available expression data, develop consensus signatures and the ability to compare gene signatures from new experiments to the analysed data to facilitate meta-analysis. AVAILABILITY AND IMPLEMENTATION: The SkeletalVis data portal is freely accessible at http://phenome.manchester.ac.uk. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

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.

Notch signaling during chondrogenesis of human bone marrow stem cells.

Notch signaling is an important mechanism involved in early development which helps to determine the differentiation and fate of cells destined to form different tissues in the body. Its role in the differentiation of adult stem cells, such as those found in bone marrow is much less clear. As there is great interest in the potential of human bone marrow stem cells (hMSC) as a source of cells for the repair of articular cartilage and other tissues, it is important to understand if Notch signaling promotes or suppresses differentiation. Using primary human bone marrow stem cells (hMSC) in 3D cell aggregate culture a new study has investigated the expression of the canonical Notch pathway genes during chondrogenesis and showed that the Notch ligand, Jagged1 (JAG1) sharply increased in expression peaking early in differentiation. A Notch target gene, HEY1, was also expressed with a temporal profile, which closely followed the expression of JAG1 and this preceded the rise in type II collagen expression that characterized chondrogenesis. The JAG1 mediated Notch signaling was shown with a Notch inhibitor (DAPT) to be necessary for chondrogenesis, as inhibition days 0-14, or just days 0-5, blocked chondrogenesis, whereas Notch inhibition days 5-14 did not. In further experiments Notch signaling was shown to be critical for full chondrogenesis, as adenoviral hJAG1 transduction of hMSCs, which caused continuous expression of JAG1 and sustained Notch signaling, completely blocked chondrogenesis. In these cultures there was inhibited production of extracellular matrix and failure to differentiate was interpreted as the retention of the hMSC in a pre-chondrogenic state. The results in this study thus showed that JAG1 mediated Notch signaling in hMSC was necessary to initiate chondrogenesis, but must be switched off for chondrogenesis to proceed and it will be important to establish if this is a mechanism common to all chondrocyte differentiation.

Chondrogenic differentiation of human bone marrow stem cells in transwell cultures: generation of scaffold-free cartilage.

Human bone marrow stem cells (hMSCs) have been shown to differentiate in vitro into a number of cell lineages and are a potential autologous cell source for the repair and replacement of damaged and diseased musculoskeletal tissues. hMSC differentiation into chondrocytes has been described in high-density cell pellets cultured with specific growth and differentiation factors. We now describe how culture of hMSCs as a shallow multicellular layer on a permeable membrane over 2-4 weeks resulted in a much more efficient formation of cartilaginous tissue than in established chondrogenic assays. In this format, the hMSCs differentiated in 14 days to produce translucent, flexible discs, 6 mm in diameter by 0.8-1 mm in thickness from 0.5 x 10(6) cells. The discs contained an extensive cartilage-like extracellular matrix (ECM), with more than 50% greater proteoglycan content per cell than control hMSCs differentiated in standard cell pellet cultures. The disc constructs were also enriched in the cartilage-specific collagen II, and this was more homogeneously distributed than in cell pellet cultures. The expression of cartilage matrix genes for collagen type II and aggrecan was enhanced in disc cultures, but improved matrix production was not accompanied by increased expression of the transcription factors SOX9, L-SOX5, and SOX6. The fast continuous growth of cartilage ECM in these cultures up to 4 weeks appeared to result from the geometry of the construct and the efficient delivery of nutrients to the cells. Scaffold-free growth of cartilage in this format will provide a valuable experimental system for both experimental and potential clinical studies.