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Funder: Biotechnology and Biological Sciences Research Council; Id: http://dx.doi.org/10.13039/501100000268, Funder: Versus Arthritis; Id: http://dx.doi.org/10.13039/501100012041, Lipids play a crucial role in signaling and metabolism, regulating the development and maintenance of the skeleton. Membrane lipids have been hypothesized to act as intermediates upstream of orphan phosphatase 1 (PHOSPHO1), a major contributor to phosphate generation required for bone mineralization. Here, we spatially resolve the lipid atlas of the healthy mouse knee and demonstrate the effects of PHOSPHO1 ablation on the growth plate lipidome. Lipids spanning 17 subclasses were mapped across the knee joints of healthy juvenile and adult mice using matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI-IMS), with annotation supported by shotgun lipidomics. Multivariate analysis identified 96 and 80 lipid ions with differential abundances across joint tissues in juvenile and adult mice, respectively. In both ages, marrow was enriched in phospholipid platelet activating factors (PAFs) and related metabolites, cortical bone had a low lipid content, whereas lysophospholipids were strikingly enriched in the growth plate, an active site of mineralization and PHOSPHO1 activity. Spatially-resolved profiling of PHOSPHO1-knockout (KO) mice across the resting, proliferating, and hypertrophic growth plate zones revealed 272, 306, and 296 significantly upregulated, and 155, 220, and 190 significantly downregulated features, respectively, relative to wild-type (WT) controls. Of note, phosphatidylcholine, lysophosphatidylcholine, sphingomyelin, lysophosphatidylethanolamine, and phosphatidylethanolamine derived lipid ions were upregulated in PHOSPHO1-KO versus WT. Our imaging pipeline has established a spatially-resolved lipid signature of joint tissues and has demonstrated that PHOSPHO1 ablation significantly alters the growth plate lipidome, highlighting an essential role of the PHOSPHO1-mediated membrane phospholipid metabolism in lipid and bone homeostasis. © 2023 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

The heterogeneity of bone marrow stromal cells (BMSCs) has been revealed more in recent years through the advent of single cell RNA sequencing. However, protein level characterisation is likely to provide a deeper understanding of the functions of individual subsets and may reveal insights into the co-ordination of the cell phenotype maintaining niche.Here, by analysing heterogeneity in BMSC populations using human stromal cell lines to model extremes of cell morphology and migration characteristics, we identified plastic cell phenotypes that can be modified through secreted proteins. Transfer of secreted signals from a differentiation-competent stem cell phenotype was able to stimulate migration in a slow-moving stromal cell, observed via label-free ptychography. Subsequent untargeted proteomic interrogation of the secreted factors from these cell lines identified a highly significant enrichment of extracellular matrix (ECM) protein production by the differentiation-competent cells compared to non-stem cells. The most highly enriched proteins, aggrecan and periostin, were identified on the endosteal surfaces of mouse and human bone, underlying CD271+ stromal cells in the latter, indicating that they may represent key non-cellular niche-components important for cell maintenance and phenotype. ECM from stem cells was further capable of enhancing migration in non-stem cells in a focal adhesion kinase-dependent manner.Overall, we demonstrate the importance of the ECM in co-ordination of cellular phenotype and highlight how non-cellular components of the BMSC niche may provide insights into the role of BMSCs in health and disease.

Protein N-termini provide uniquely reactive motifs for single site protein modification. Though a number of reactions have been developed to target this site, the selectivity, generality, and stability of the conjugates formed has not been studied. We have therefore undertaken a comprehensive comparative study of the most promising methods for N-terminal protein modification, and find that there is no ‘one size fits all’ approach, necessitating reagent screening for a particular protein or application. Moreover, we observed limited stability in all cases, leading to a need for continued innovation and development in the bioconjugation field.

Bone marrow mesenchymal stem/stromal cells (MSCs) maintain bone homeostasis and repair through the ability to expand in response to mitotic stimuli and differentiate into skeletal lineages. Signalling mechanisms that enable precise control of MSC function remain unclear. Here we report that by initially examining differences in signalling pathway expression profiles of individual MSC clones, we identified a previously unrecognised signalling mechanism regulated by epidermal growth factor (EGF) in primary human MSCs. We demonstrate that EGF is able to activate β-catenin, a key component of the canonical Wnt signalling pathway. EGF is able to induce nuclear translocation of β-catenin in human MSCs but does not drive expression of Wnt target genes or T cell factor (TCF) activity in MSC reporter cell lines. Using an efficient Design of Experiments (DoE) statistical analysis, with different combinations and concentrations of EGF and Wnt ligands, we were able to confirm that EGF does not influence the Wnt/β-catenin pathway in MSCs. We show that the effects of EGF on MSCs are temporally regulated to initiate early “classical” EGF signalling mechanisms (e.g via mitogen activated protein kinase) with delayed activation of β-catenin. By RNA-sequencing, we identified gene sets that were exclusively regulated by the EGF/β-catenin pathway, which were distinct from classical EGF-regulated genes. However, subsets of classical EGF gene targets were significantly influenced by EGF/β-catenin activation. These signalling pathways cooperate to enable EGF-mediated proliferation of MSCs by alleviating the suppression of cell cycle pathways induced by classical EGF signalling., Highlights • Epidermal growth factor (EGF) controls mesenchymal stem cell (MSC) proliferation. • EGF signals through β-catenin in MSCs but not in related fibroblastic cells. • Classical EGF and EGF/β-catenin cooperatively regulate distinct gene sets in MSCs. • EGF/β-catenin enables MSC proliferation by alleviating cell cycle suppression.

Glycans are inherently heterogeneous, yet glycosylation is essential in eukaryotes, and glycans show characteristic cell type-dependent distributions. By using an immortalized human mesenchymal stromal cell (MSC) line model, we show that both N- and O-glycan processing in the Golgi functionally modulates early steps of osteogenic differentiation. We found that inhibiting O-glycan processing in the Golgi prior to the start of osteogenesis inhibited the mineralization capacity of the formed osteoblasts 3 weeks later. In contrast, inhibition of N-glycan processing in MSCs altered differentiation to enhance the mineralization capacity of the osteoblasts. The effect of N-glycans on MSC differentiation was mediated by the phosphoinositide-3-kinase (PI3K)/Akt pathway owing to reduced Akt phosphorylation. Interestingly, by inhibiting PI3K during the first 2 days of osteogenesis, we were able to phenocopy the effect of inhibiting N-glycan processing. Thus, glycan processing provides another layer of regulation that can modulate the functional outcome of differentiation. Glycan processing can thereby offer a novel set of targets for many therapeutically attractive processes., Summary: Both N- and O-glycan processing modulate MSC differentiation early during osteogenesis to influence mineral formation. Inhibition of N-glycan processing increases mineralization.

A method has been developed for release/isolation of O-glycans from glycoproteins in whole cell lysates for mass spectrometric analysis. Cells are lysed in SDS, which is then exchanged for urea and ammonium bicarbonate in a centrifugal filter, before treating with NH

Mesenchymal stem cells (MSCs) are multipotent stem cells, found in the bone-marrow and other adult tissues, which give rise to various cell lineages. Although MSCs are biologically important, and may have widespread therapeutic potential, they are not well-characterised, particularly in terms of their cell surface receptors and in vivo phenotype. We aimed to develop a three-dimensional (3-D) MSC in vitro model, in order to understand the factors involved in the regulation of lineage specification routes. A suitable model, which replicates the MSC microenvironment as accurately as possible, will allow more-detailed investigations into the phenotype of the cells. Our MSC spheroids appear to have an enhanced mesenchymal differentiation compared to two-dimensional MSC monolayers. With this in vitro system, it is possible to perform real-time analysis of cellular differentiation status. MSC spheroids may also be amenable for use in high-throughput assays. A more-recent research project aims to generate knockout micro-tissues, based on human 3-D MSCs, as an alternative to animal studies.

Background: Lin28 proteins are post‐transcriptional regulators of gene expression with multiple roles in development and the regulation of pluripotency in stem cells. Much attention has focussed on Lin28 proteins as negative regulators of let‐7 miRNA biogenesis; a function that is conserved in several animal groups and in multiple processes. However, there is increasing evidence that Lin28 proteins have additional roles, distinct from regulation of let‐7 abundance. We have previously demonstrated that lin28 proteins have functions associated with the regulation of early cell lineage specification in Xenopus embryos, independent of a lin28/let‐7 regulatory axis. However, the nature of lin28 targets in Xenopus development remains obscure. Results: Here, we show that mir‐17∼92 and mir‐106∼363 cluster miRNAs are down‐regulated in response to lin28 knockdown, and RNAs from these clusters are co‐expressed with lin28 genes during germ layer specification. Mature miRNAs derived from pre‐mir‐363 are most sensitive to lin28 inhibition. We demonstrate that lin28a binds to the terminal loop of pre‐mir‐363 with an affinity similar to that of let‐7, and that this high affinity interaction requires to conserved a GGAG motif. Conclusions: Our data suggest a novel function for amphibian lin28 proteins as positive regulators of mir‐17∼92 family miRNAs. Developmental Dynamics 245:34–46, 2016. © 2015 Wiley Periodicals, Inc., Key Findings We show that mir‐17∼92 and mir‐106∼363 cluster miRNAs are down regulated in response to lin28 knockdown in Xenopus embryos.We demonstrate that lin28a binds to the terminal loop of pre‐mir‐363 and this interaction requires a conserved a GGAG motif. Our data suggest a novel function for amphibian lin28 proteins as positive regulators of mir‐17∼92 family miRNAs.

The field of tissue engineering has developed rapidly over the last few decades and is of great relevance to musculoskeletal therapy and intervention. Tissue engineering strategies are often considered in a simplified form in terms of cells, scaffolds, and additional factors, although it should be noted that successful translation of such a strategy is more complex. There are many variations of usage and combination and it is not necessary for all three to be provided by the proposed treatment. However, the regenerative approach must produce both the quantity and quality of target tissue at the level of the cell, matrix, and environment. Moreover, the regenerated tissue must interact with the host tissue with a seamless biological and functional interface. Tissue engineering, regenerative medicine, and cell therapies have developed significantly over the last few decades and are applicable to a wide range of musculoskeletal applications. Many strategies have been identified that would potentially benefit patients but the future will require successful translation from the laboratory into clinical practice. It is important to identify clinical targets where there is both clinical need and an informed view that the approach is likely to be successful.

A population of multipotent stromal cells exists within bone marrow and other adult tissues, which is able to differentiate into different skeletal tissues such as bone, cartilage and fat. These cells are frequently referred to as mesenchymal stem cells (MSCs) and offer significant therapeutic potential, particularly in orthopaedic applications, but may also have broader roles in regenerative medicine, cancer treatment, as anti-inflammatories, immunosuppressives and vehicles for gene/protein therapy. Much attention has focused on understanding MSC biology and the regulation of differentiation to help realise these clinical aspirations. Here we review some of the key molecular determinants of MSC function, with an emphasis on transcription factor control and the cell-cell signalling pathways that regulate MSC differentiation. The source information comes from a range of different models, including isolated human MSC cultures, animal-derived MSC-like cell lines, animal models and skeletal developmental processes to provide a wide-angled overview of the important players in MSC biology and tri-lineage specification.

In recent years, transcriptomics and proteomics have provided us with a great deal of information about the expression profiles of various cell types and how these change under different conditions. Stem cell research is one area where this has had a major impact by providing an insight into events at the molecular level that control stem cell growth and differentiation. This includes mesenchymal stem cell (MSC) biology where knowledge about the mechanisms governing differentiation is vital for the development of future therapeutic strategies. Although there is still much to learn, we are starting to build up a picture of the main events in these differentiation processes. This review will discuss control of MSC differentiation at the transcriptional level. Not all the factors which have been shown to play a role in lineage-specific mesenchymal differentiation can be covered here. Instead, we will focus specifically on the key factors that contribute to the regulation of osteogenesis, adipogenesis, and chondrogenesis.In den vergangenen Jahren bescherte uns die Transkriptomik und Proteomik eine Menge an Informationen über die Expressionsprofile von verschiedenen Zelltypen und darüber, wie sich diese unter verschiedenen Bedingungen ändern. Stammzellforschung ist ein Bereich, in dem dies einen großen Einfluss hatte, weil dort Einblicke erzielt wurden, wie Stammzellwachstum und -differenzie-rung auf molekularer Ebene kontrolliert werden. Das schließt auch die Biologie der mesenchymalen Stammzellen (MSCs) mit ein, wo das Wissen über die Mechanismen, die die Differenzierung steuern, entscheidend ist für die Entwicklung zukünftiger therapeutischer Strategien. Obwohl immer noch vieles nicht bekannt ist, beginnt man die Hauptereignisse innerhalb der Differenzie-rungsprozess zu verstehen. Die Kontrolle der MSC-Differenzierung auf Transkriptionsebene bildet den Schwerpunkt der vorliegenden Übersicht, wenn auch nicht alle Faktoren, die bei der abstammungsspezifischen mesenchymalen Differenzierung eine Rolle spielen, hier dargestellt werden können. Stattdessen wollen wir uns besonders auf die Schlüsselfaktoren, die zur Regulation der Osteogenese, der Adipogenese und der Chondrogenese beitragen, beschränken.

Musculoskeletal disease is a leading cause of morbidity across the world, associated with pain, immobility, deformities, and in some cases, death. Other factors affecting quality of life include lack of independence, an inability to perform routine tasks, and reduced social interaction. Disease prevalence increases with age and we are living longer. In the coming years, the incidence of disorders affecting the skeleton will rise, causing huge healthcare and socioeconomic burden. Current treatments are typically restricted to pain management followed by end-stage total joint replacement. Cell-based therapies are an appealing biological option in orthopedics, as they may provide long-lasting restoration of skeletal tissue function by exploiting the intrinsic stem cell-like capacity of mesenchymal stromal cells (MSCs) to differentiate into bone and cartilage. Here, we review recent data on the use of MSCs in orthopedics, focusing on clinical trials and discussing the advances made as well as the hurdles to overcome.