Osteoarthritis (OA) is a rheumatic disease leading to chronic pain and disability with no effective treatment available. Recently, allogeneic human mesenchymal stromal/stem cells (MSC) entered clinical trials as a novel therapy for OA. Increasing evidence suggests that therapeutic efficacy of MSC depends on paracrine signalling. Here we investigated the role of extracellular vesicles (EVs) secreted by human bone marrow derived MSC (BMMSC) in human OA cartilage repair.Methods: To test the effect of BMMSC-EVs on OA cartilage inflammation, TNF-alpha-stimulated OA chondrocyte monolayer cultures were treated with BMMSC-EVs and pro-inflammatory gene expression was measured by qRT-PCR after 48 h. To assess the impact of BMMSC-EVs on cartilage regeneration, BMMSC-EVs were added to the regeneration cultures of human OA chondrocytes, which were analyzed after 4 weeks for glycosaminoglycan content by 1,9-dimethylmethylene blue (DMMB) assay. Furthermore, paraffin sections of the regenerated tissue were stained for proteoglycans (safranin-O) and type II collagen (immunostaining).Results: We show that BMMSC-EVs inhibit the adverse effects of inflammatory mediators on cartilage homeostasis. When co-cultured with OA chondrocytes, BMMSC-EVs abrogated the TNF-alpha-mediated upregulation of COX2 and pro-inflammatory interleukins and inhibited TNF-alpha-induced collagenase activity. BMMSC-EVs also promoted cartilage regeneration in vitro. Addition of BMMSC-EVs to cultures of chondrocytes isolated from OA patients stimulated production of proteoglycans and type II collagen by these cells.Conclusion: Our data demonstrate that BMMSC-EVs can be important mediators of cartilage repair and hold great promise as a novel therapeutic for cartilage regeneration and osteoarthritis.

Loss of SMAD4 from colorectal cancer cells causes BMP signaling to switch from tumor suppressive to metastasis promoting. Concurrent loss of SMAD4 and normal expression of BMP receptors in colorectal tumors was associated with reduced survival times of patients. Reagents that interfere with SMAD4-independent BMP signaling, such as ROCK inhibitors, might be developed as therapeutics for CRC.

Correlative light and electron microscopy (CLEM) is a unique method for investigating biological structure-function relations. With CLEM protein distributions visualized in fluorescence can be mapped onto the cellular ultrastructure measured with electron microscopy. Widespread application of correlative microscopy is hampered by elaborate experimental procedures related foremost to retrieving regions of interest in both modalities and/or compromises in integrated approaches. We present a novel approach to correlative microscopy, in which a high numerical aperture epi-fluorescence microscope and a scanning electron microscope illuminate the same area of a sample at the same time. This removes the need for retrieval of regions of interest leading to a drastic reduction of inspection times and the possibility for quantitative investigations of large areas and datasets with correlative microscopy. We demonstrate Simultaneous CLEM (SCLEM) analyzing cell-cell connections and membrane protrusions in whole uncoated colon adenocarcinoma cell line cells stained for actin and cortactin with AlexaFluor488. SCLEM imaging of coverglass-mounted tissue sections with both electron-dense and fluorescence staining is also shown.

Developments in microscopy have been instrumental to progress in the life sciences, and many new techniques have been introduced and led to new discoveries throughout the last century. A wide and diverse range of methodologies is now available, including electron microscopy, atomic force microscopy, magnetic resonance imaging, small-angle x-ray scattering and multiple super-resolution fluorescence techniques, and each of these methods provides valuable read-outs to meet the demands set by the samples under study. Yet, the investigation of cell development requires a multi-parametric approach to address both the structure and spatio-temporal organization of organelles, and also the transduction of chemical signals and forces involved in cell–cell interactions. Although the microscopy technologies for observing each of these characteristics are well developed, none of them can offer read-out of all characteristics simultaneously, which limits the information content of a measurement. For example, while electron microscopy is able to disclose the structural layout of cells and the macromolecular arrangement of proteins, it cannot directly follow dynamics in living cells. The latter can be achieved with fluorescence microscopy which, however, requires labelling and lacks spatial resolution. A remedy is to combine and correlate different readouts from the same specimen, which opens new avenues to understand structure–function relations in biomedical research. At the same time, such correlative approaches pose new challenges concerning sample preparation, instrument stability, region of interest retrieval, and data analysis. Because the field of correlative microscopy is relatively young, the capabilities of the various approaches have yet to be fully explored, and uncertainties remain when considering the best choice of strategy and workflow for the correlative experiment. With this in mind, the Journal of Physics D: Applied Physics presents a special roadmap on the correlative microscopy techniques, giving a comprehensive overview from various leading scientists in this field, via a collection of multiple short viewpoints.

Multisubunit tethering complexes (MTCs) are multitasking hubs that form a link between membrane fusion, organelle motility and signaling. CORVET, CHEVI and HOPS are MTCs of the endo-lysosomal system. They regulate the major membrane flows required for endocytosis, lysosome biogenesis, autophagy and phagocytosis. In addition, individual subunits control complex-independent transport of specific cargoes and exert functions beyond tethering, such as attachment to microtubules and SNARE activation. Mutations in CHEVI subunits lead to arthrogryposis, renal dysfunction and cholestasis (ARC) syndrome, while defects in CORVET and, particularly, HOPS are associated with neurodegeneration, pigmentation disorders, liver malfunction and various forms of cancer. Diseases and phenotypes, however, vary per affected subunit and a concise overview of MTC protein function and associated human pathologies is currently lacking. Here, we provide an integrated overview on the cellular functions and pathological defects associated with CORVET, CHEVI or HOPS proteins, both with regard to their complexes and as individual subunits. The combination of these data provides novel insights into how mutations in endo-lysosomal proteins lead to human pathologies.

Live-cell correlative light-electron microscopy (live-cell-CLEM) integrates live movies with the corresponding electron microscopy (EM) image, but a major challenge is to relate the dynamic characteristics of single organelles to their 3-dimensional (3D) ultrastructure. Here, we introduce focused ion beam scanning electron microscopy (FIB-SEM) in a modular live-cell-CLEM pipeline for a single organelle CLEM. We transfected cells with lysosomal-associated membrane protein 1-green fluorescent protein (LAMP-1-GFP), analyzed the dynamics of individual GFP-positive spots, and correlated these to their corresponding fine-architecture and immediate cellular environment. By FIB-SEM we quantitatively assessed morphological characteristics, like number of intraluminal vesicles and contact sites with endoplasmic reticulum and mitochondria. Hence, we present a novel way to integrate multiple parameters of subcellular dynamics and architecture onto a single organelle, which is relevant to address biological questions related to membrane trafficking, organelle biogenesis and positioning. Furthermore, by using CLEM to select regions of interest, our method allows for targeted FIB-SEM, which significantly reduces time required for image acquisition and data processing.

Bone formation (osteogenesis) is a complex process in which cellular differentiation and the generation of a mineralized organic matrix are synchronized to produce a hybrid hierarchical architecture. To study the mechanisms of osteogenesis in health and disease, there is a great need for functional model systems that capture in parallel, both cellular and matrix formation processes. Stem cell‐based organoids are promising as functional, self‐organizing 3D in vitro models for studying the physiology and pathology of various tissues. However, for human bone, no such functional model system is yet available. This study reports the in vitro differentiation of human bone marrow stromal cells into a functional 3D self‐organizing co‐culture of osteoblasts and osteocytes, creating an organoid for early stage bone (woven bone) formation. It demonstrates the formation of an organoid where osteocytes are embedded within the collagen matrix that is produced by the osteoblasts and mineralized under biological control. Alike in in vivo osteocytes, the embedded osteocytes show network formation and communication via expression of sclerostin. The current system forms the most complete 3D living in vitro model system to investigate osteogenesis, both in physiological and pathological situations, as well as under the influence of external triggers (mechanical stimulation, drug administration).

Differential signalling of the WNT and Notch pathways regulates proliferation and differentiation of Lgr5 + cryptbased columnar cells (CBCs) into all cell lineages of the intestine. We have recently shown that high mitochondrial activity in CBCs is key in maintaining stem cell function. Interestingly, while high mitochondrial activity drives CBCs, it is reduced in the adjacent secretory Paneth cells (PCs). This observation implies that during differentiation towards PCs, CBCs undergo a metabolic rewiring involving downregulation of mitochondrial number and activity, through a hitherto unknown mechanism. Here we demonstrate, using intestinal organoids that FoxO transcription factors and Notch signalling functionally interact in determining CBC cell fate. In agreement with the organoid data, combined Foxo1 and 3 deletion in mice increases PC number in the intestine. Importantly, we show that FOXO and Notch signalling converge onto regulation of mitochondrial fission, which in turn provokes stem cell differentiation into the secretory types; Goblet cells and PCs. Finally, mapping intestinal stem cell differentiation based on pseudotime computation of scRNA-seq data further supports the role of FOXO, Notch and mitochondria in determining secretory differentiation. This shows that mitochondria is not only a discriminatory hallmark of CBCs and PCs, but that its status actively determines lineage commitment during differentiation. Together, our work describes a new signalling-metabolic axis in stem cell differentiation and highlights the importance of mitochondria in determining cell fate.