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ObjectivesIt has been suggested that the lysosomal recycling process called macro-autophagy plays a role in osteoarthritis development. We thus decided to genetically ablate the autophagy-indispensable Atg5 gene specifically in chondrocytes and analyse the development of osteoarthritis upon aging and in a post-traumatic model.MethodsMice lacking the Atg5 gene in their chondrocytes (Atg5cKO) were generated by crossing Atg5-floxed mice with transgenic mice that expressed cre recombinase driven by the collagen type 2 promoter. Animals were analysed at the age of 2, 6 and 12 months for age-related osteoarthritis or underwent mini-open partial medial meniscectomy at 2 months of age and were analysed 1 or 2 months after surgery. We evaluated osteoarthritis using the Osteoarthritis Research Society International (OARSI) scoring on safranin-O-stained samples. Cell death was evaluated by terminal deoxy-nucleotidyl-transferase-mediated deoxy-UTP nick end labelling (TUNEL) and by immunostaining of cleaved caspases.ResultsWe observed the development of osteoarthritis in Atg5cKO mice with aging including fibrillation and loss of proteoglycans, which was particularly severe in males. The ablation of Atg5 was associated with an increased cell death as assessed by TUNEL, cleaved caspase 3 and cleaved caspase 9. Surprisingly, no difference in the development of post-traumatic osteoarthritis was observed between Atg5cKO and control mice.ConclusionsAutophagy protects from age-related osteoarthritis by facilitating chondrocyte survival.

Established cell culture systems have failed to accurately recapitulate key features of terminal erythroid maturation, hampering our ability to in vitro model and treat diseases with impaired erythropoiesis such as myelodysplastic syndromes with ring sideroblasts (MDS-RS). We developed an efficient and robust three-dimensional (3D) scaffold culture model supporting terminal erythroid differentiation from both mononuclear (MNC) or CD34+-enriched primary bone marrow cells from healthy donors and MDS-RS patients. While CD34+ cells did not proliferate beyond two weeks in 2D suspension cultures, the 3D scaffolds supported CD34+ and MNC erythroid proliferation over four weeks demonstrating the importance of the 3D environment. CD34+ cells cultured in 3D facilitated the highest expansion and maturation of erythroid cells, including generation of erythroblastic islands and enucleated erythrocytes, while MNCs supported multi-lineage hemopoietic differentiation and cytokine secretion relevant for MDS-RS. Importantly, MDS-RS 3D-cultures supported de novo generation of ring sideroblasts and maintenance of the mutated clone. The 3D cultures effectively model a clonal disease characterized by terminal erythroid failure and can be used to assess therapeutic compounds.

Reef-building corals have a variety of green fluorescent protein (GFP)-like proteins, also known as fluorescent proteins (FPs). These proteins have broad spectral properties covering most of the visible spectrum, with fluorophores fluorescing from cyan to red. However, the role of FPs is still a topic of debate and requires further investigation, particularly in the direction of mapping these FPs within the coral tissue and describing their cell- and tissue-level distributions. This study applied confocal laser scanning microscopy (CLSM) to investigate species-specific differences in the distribution of FPs in three coral taxa ( Stylophora sp., Acropora sp., Echinopora sp.), combined with their photoacclimation response and that of associated symbiotic algae to light gradients in a controlled aquarium experiment. CLSM produced high-resolution images that enabled the identification of different FPs, their tissue distribution and quantification of their fluorescence intensity, as well as quantification of symbiont chlorophyll a (chl- a ) fluorescence. Emission scans revealed three emission peaks between 490 - 501 nm (cyan, CFPs), 510 - 515 nm (green, GFPs), and 679 nm (chl- a fluorescence signal; Fchl) shared by all three studied species. The distribution of GFPs in Stylophora was concentrated in the intermesenterial muscle bands of the polyp, whereas CFPs were typically located at the tips of the tentacles. In contrast, Acropora and Echinopora exhibited agglomeration of CFPs and GFPs primarily in the epidermis. In general, species-specific differences in FP distribution remained unaltered during the experiment. However, linear regression models showed a significant negative relationship between CFP fluorescence intensity and light irradiance in Stylophora , whereas Echinopora exhibited a negative relation between chlorophyll fluorescence (Fchl) and light. In summary, the CLSM methodology provided a high-resolution tool to study coral FP patterns and symbiont response to irradiance, revealing ecophysiological differences among coral species at the tissue and cellular levels. CLSM has the potential to elucidate the intricacies of coral photobiology within the natural environment and to discern their adaptive responses in situ .

Pleiotrophin (PTN) is a growth factor present in the extracellular matrix of the growth plate during bone development and in the callus during bone healing. Bone healing is a complicated process that recapitulates endochondral bone development and involves many cell types. Among those cells, mesenchymal stromal cells (MSC) are able to differentiate toward chondrogenic and osteoblastic lineages. We aimed to determine PTN effects on differentiation properties of human bone marrow stromal cells (hBMSC) under chondrogenic induction using histological analysis and quantitative reverse transcription polymerase chain reaction. PTN dramatically potentiated chondrogenic differentiation as indicated by a strong increase of collagen 2 protein, and cartilage-related gene expression. Moreover, PTN increased transcription of hypertrophic chondrocyte markers such as MMP13, collagen 10 and alkaline phosphatase and enhanced calcification and the content of collagen 10 protein. These effects are dependent on PTN receptors signaling and PI3 K pathway activation. These data suggest a new role of PTN in bone regeneration as an inducer of hypertrophy during chondrogenic differentiation of hBMSC.

Longitudinal bone growth in children is sustained by growth plates, narrow discs of cartilage that provide a continuous supply of chondrocytes for endochondral ossification 1 . However, it remains unknown how this supply is maintained throughout childhood growth. Chondroprogenitors in the resting zone are thought to be gradually consumed as they supply cells for longitudinal growth 1 , 2 , but this model has never been proved. Here, using clonal genetic tracing with multicolour reporters and functional perturbations, we demonstrate that longitudinal growth during the fetal and neonatal periods involves depletion of chondroprogenitors, whereas later in life, coinciding with the formation of the secondary ossification centre, chondroprogenitors acquire the capacity for self-renewal, resulting in the formation of large, stable monoclonal columns of chondrocytes. Simultaneously, chondroprogenitors begin to express stem cell markers and undergo symmetric cell division. Regulation of the pool of self-renewing progenitors involves the hedgehog and mammalian target of rapamycin complex 1 (mTORC1) signalling pathways. Our findings indicate that a stem cell niche develops postnatally in the epiphyseal growth plate, which provides a continuous supply of chondrocytes over a prolonged period. Clonal genetic tracing is used to demonstrate that, in mice, longitudinal bone growth during fetal and neonatal periods relies on the gradual consumption of chondroprogenitors, whereas in adults, a stem cell niche is formed allowing renewing of chondroprogenitors and leading to formation of large, stable monoclonal columns of chondrocytes.

Characterization of the progression of cellular states during human embryogenesis can provide insights into the origin of pediatric diseases. We examined the transcriptional states of neural crest– and mesoderm-derived lineages differentiating into adrenal glands, kidneys, endothelium and hematopoietic tissue between post-conception weeks 6 and 14 of human development. Our results reveal transitions connecting the intermediate mesoderm and progenitors of organ primordia, the hematopoietic system and endothelial subtypes. Unexpectedly, by using a combination of single-cell transcriptomics and lineage tracing, we found that intra-adrenal sympathoblasts at that stage are directly derived from nerve-associated Schwann cell precursors, similarly to local chromaffin cells, whereas the majority of extra-adrenal sympathoblasts arise from the migratory neural crest. In humans, this process persists during several weeks of development within the large intra-adrenal ganglia-like structures, which may also serve as reservoirs of originating cells in neuroblastoma. Single-cell transcriptome profiling of human embryonic sympathoadrenal tissues identifies developmental transitions and suggests that intra-adrenal sympathoblasts arising from Schwann cell precursors are a potential neuroblastoma cell of origin.

Electrospinning coupled with electrospraying provides a straightforward and robust route toward promising electrospun biocomposite scaffolds for bone tissue engineering. In this comparative investigation, four types of poly(3-hydroxybutyrate) (PHB)-based nanofibrous scaffolds were produced by electrospinning a PHB solution, a PHB/gelatin (GEL) mixture or a PHB/GEL/nHAs (hydroxyapatite nanoparticles) mixed solution, and by electrospinning a PHB/GEL solution and electrospraying a nHA dispersion simultaneously. SEM and TEM analyses demonstrated that the electrospun nHA-blended framework contained a majority of nHAs trapped within the constitutive fibers, whereas the electrospinning-electrospraying combination afforded fibers with a rough surface largely covered by the bioceramic. Structural and morphological characterizations were completed by FTIR, mercury intrusion porosimetry, and contact angle measurements. Furthermore, an in vitro investigation of human mesenchymal stromal cell (hMSC) adhesion and proliferation properties showed a faster cell development on gelatin-containing scaffolds. More interestingly, a long-term investigation of hMSC osteoblastic differentiation over 21 days indicate that hMSCs seeded onto the nHA-sprayed scaffold developed a significantly higher level of alkaline phosphatase activity, as well as a higher matrix biomineralization rate through the staining of the generated calcium deposits: the fiber surface deposition of nHAs by electrospraying enabled their direct exposure to hMSCs for an efficient transmission of the bioceramic osteoinductive and osteoconductive properties, producing a suitable biocomposite scaffold for bone tissue regeneration.

In this study we use comparative genomics to uncover a gene with uncharacterized function ( 1700011H14Rik/C14orf105/CCDC198 ), which we hereby name FAME (Factor Associated with Metabolism and Energy). We observe that FAME shows an unusually high evolutionary divergence in birds and mammals. Through the comparison of single nucleotide polymorphisms, we identify gene flow of FAME from Neandertals into modern humans. We conduct knockout experiments on animals and observe altered body weight and decreased energy expenditure in Fame knockout animals, corresponding to genome-wide association studies linking FAME with higher body mass index in humans. Gene expression and subcellular localization analyses reveal that FAME is a membrane-bound protein enriched in the kidneys. Although the gene knockout results in structurally normal kidneys, we detect higher albumin in urine and lowered ferritin in the blood. Through experimental validation, we confirm interactions between FAME and ferritin and show co-localization in vesicular and plasma membranes. The human genome still contains numerous uncharacterized genes. Here, the authors identify a fast evolving Factor associated with Metabolism and Energy (FAME) that is associated with altered body weight, energy expenditure, and metabolism and study its function in knockout mouse models.

Tissue engineering approaches to stimulate bone formation currently combine bioactive scaffolds with osteocompetent human mesenchymal stem cells (hMSC). Moreover, osteogenic and angiogenic factors are required to promote differentiation and survival of hMSC through improved vascularization through the damaged extracellular matrix (ECM). Glycosaminoglycans (GAGs) are ECM compounds acting as modulators of heparin-binding protein activities during bone development and regenerative processes. GAG mimetics have been proposed as ECM stabilizers and were previously described for their positive effects on bone formation and angiogenesis after local treatment. Here, we developed a strategy associating the GAG mimetic [OTR 4120 ] with bone substitutes to optimize stem cell-based therapeutic products. We showed that [OTR 4120 ] was able to potentiate proliferation, migration, and osteogenic differentiation of hMSC in vitro . Its link to tricalcium phosphate/hydroxyapatite scaffolds improved their colonization by hMSC. Surprisingly, when these combinations were tested in an ectopic model of bone formation in immunodeficient mice, the GAG mimetics inhibit bone formation induced by hMSC and promoted an osteoclastic activity. Moreover, the inflammatory response was modulated, and the peri-implant vascularization stimulated. All together, these findings further support the ability of GAG mimetics to organize the local ECM to coordinate the host response toward the implanted biomaterial, and to inhibit the abnormal bone formation process on a subcutaneous ectopic site.