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Lung cancer is among the most clinically challenging tumors because of its aggressive proliferation, metastasis, and the presence of cancer stem cells (CSCs). Natural bioactive substances have been used for cancer prevention, and, in particular, resveratrol (RSV), a stilbene-based compound with wide biological properties, has been proposed for chemoprevention. Its lesser-known analogue 4,4'-dihydroxy-trans-stilbene (DHS) has demonstrated superior activity both in cell-based assays and in mouse and zebrafish in vivo models. The present study analyzed the effects of DHS and RSV on A549 lung cancer cells, with a particular focus on stemness features and CSCs, isolated by sorting of the side population (SP). The results show that both stilbenes, especially DHS, strongly inhibited cell cycle progression. A reduction in the S phase was induced by DHS, whereas an increase in this phase was obtained with RSV. In addition, 50% reductions in the clonogenicity and soft agar colony formation were observed with the DHS treatment only. Finally, both stilbenes, especially DHS, reduced stemness marker expression in A549 cells and their sorted SP fraction. Spheroid formation, higher in SP cells than in the main population (MP), was significantly reduced after pretreatment with DHS, which was found to decrease SOX2 levels more than RSV. These findings indicate that stilbenes, and particularly DHS, affect stemness features of A549 cells and the SP fraction, suggesting their potential utility as anticancer agents, either alone or combined with chemotherapeutic drugs.

Bioresorbable chitosan scaffolds have shown potential for osteochondral repair applications. The in vivo degradation of chitosan, mediated by lysozyme and releasing glucosamine, enables progressive replacement by ingrowing tissue. Here the degradation process of a chitosan-nHA based bioresorbable scaffold was investigated for mass loss, mechanical properties and degradation products released from the scaffold when subjected to clinically relevant enzyme concentrations. The scaffold showed accelerated mass loss during the early stages of degradation but without substantial reduction in mechanical strength or structure deterioration. Although not cytotoxic, the medium in which the scaffold was degraded for over 2 weeks showed a transient decrease in mesenchymal stem cell viability, and the main degradation product (glucosamine) demonstrated a possible adverse effect on viability when added at its peak concentration. This study has implications for the design and biomedical application of chitosan scaffolds, underlining the importance of modelling degradation products to determine suitability for clinical translation., (Creative Commons Attribution license.)

Joubert syndrome (JS) is a recessively inherited congenital ataxia characterized by hypotonia, psychomotor delay, abnormal ocular movements, intellectual disability, and a peculiar cerebellar and brainstem malformation, the "molar tooth sign." Over 40 causative genes have been reported, all encoding for proteins implicated in the structure or functioning of the primary cilium, a subcellular organelle widely present in embryonic and adult tissues. In this paper, we developed an in vitro neuronal differentiation model using patient-derived induced pluripotent stem cells (iPSCs), to evaluate possible neurodevelopmental defects in JS. To this end, iPSCs from four JS patients harboring mutations in distinct JS genes (AHI1, CPLANE1, TMEM67, and CC2D2A) were differentiated alongside healthy control cells to obtain mid-hindbrain precursors and cerebellar granule cells. Differentiation was monitored over 31 days through the detection of lineage-specific marker expression by qRT-PCR, immunofluorescence, and transcriptomics analysis. All JS patient-derived iPSCs, regardless of the mutant gene, showed a similar impairment to differentiate into mid-hindbrain and cerebellar granule cells when compared to healthy controls. In addition, analysis of primary cilium count and morphology showed notable ciliary defects in all differentiating JS patient-derived iPSCs compared to controls. These results confirm that patient-derived iPSCs are an accessible and relevant in vitro model to analyze cellular phenotypes connected to the presence of JS gene mutations in a neuronal context., (© 2024. The Author(s).)

Osteochondral grafts are used for repair of focal osteochondral lesions. Autologous grafts are the gold standard treatment; however, limited graft availability and donor site morbidity restrict use. Therefore, there is a clinical need for different graft sources/materials which replicate natural cartilage function. Chitosan has been proposed for this application. The aim of this study was to assess the biomechanics and biotribology of a bioresorbable chitosan/chitosan-nano-hydroxyapatite osteochondral construct (OCC), implanted in an in vitro porcine knee experimental simulation model. The OCC implanted in different surgical positions (flush, proud and inverted) was compared to predicate grafts in current clinical use and a positive control consisting of a stainless steel graft implanted proud of the cartilage surface. After 3 h (10 800 cycles) wear simulation under a walking gait, subsidence occurred in all OCC samples irrespective of surgical positioning, but with no apparent loss of material and low meniscus wear. Half the predicate grafts exhibited delamination and scratching of the cartilage surfaces. No graft subsidence occurred in the positive controls but wear and deformation of the meniscus were apparent. Implanting a new chitosan-based OCC either optimally (flush), inverted or proud of the cartilage surface resulted in minimal wear, damage and deformation of the meniscus., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

Although phenotypically different, brown adipose tissue (BAT) and inguinal white adipose tissue (iWAT) are able to produce heat through non-shivering thermogenesis due to the presence of mitochondrial uncoupling protein 1 (UCP1). The appearance of thermogenically active beige adipocytes in iWAT is known as browning. Both brown and beige cells originate from mesenchymal stem cells (MSCs), and in culture conditions a browning response can be induced with hypothermia (i.e. 32 °C) during which nuclear leptin immunodetection was observed. The central role of leptin in regulating food intake and energy consumption is well recognised, but its importance in the browning process at the cellular level is unclear. Here, immunocytochemical analysis of MSC-derived adipocytes established nuclear localization of both leptin and leptin receptor suggesting an involvement of the leptin pathway in the browning response. In order to elucidate whether leptin modulates the expression of brown and beige adipocyte markers, BAT and iWAT samples from leptin-deficient (ob/ob) mice were analysed and exhibited reduced brown/beige marker expression compared to wild-type controls. When MSCs were isolated and differentiated into adipocytes, leptin deficiency was observed to induce a white phenotype, especially when incubated at 32 °C. These adaptations were accompanied with morphological signs of impaired adipogenic differentiation. Overall, our results indicate that leptin supports adipocyte browning and suggest a potential role for leptin in adipogenesis and browning., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Adipocytes and osteoblasts derive from a common mesenchymal progenitor present in a range of connective tissues. Differentiation of the progenitors toward the two cell lineages can be induced in vitro through well-established protocols, and leads to the appearance of lipid-laden adipocytes and osteoblasts embedded in a mineralized matrix. The formation of these two lineages in cell cultures can be monitored using lipophilic dyes such as Oil Red O and substances binding to mineral deposits such as Alizarin Red S, respectively. However, these common staining techniques require cell fixation and are thus incompatible with live analyses. Recently, alternative approaches using vital stains have allowed the dual visualization and fluorescence imaging of adipogenic and osteogenic lineages in live cultures. Here we present the concomitant analysis of cultures containing adipogenic and osteogenic cell types using live staining, combining LipidTox Red and tetracycline with NucRed nuclear counterstain for confocal imaging. This approach can be applied to visualize the kinetics and 3D structure of differentiating mesenchymal cultures over time and highlights the interaction of adipose and mineralized compartments associated with bone marrow stroma., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Lung cancer is the most prevalent cancer worldwide. Despite advances in surgery and immune-chemotherapy, the therapeutic outcome remains poor. In recent years, the anticancer properties of natural compounds, along with their low toxic side effects, have attracted the interest of researchers. Resveratrol (RSV) and many of its derivatives received particular attention for their beneficial bioactivity. Here we studied the activity of RSV and of its analogue 4,4'-dihydroxystilbene (DHS) in C57BL/6J mice bearing cancers resulting from Lung Lewis Carcinoma (LLC) cell implantation, considering tumour mass weight, angiogenesis, cell proliferation and death, autophagy, as well as characterization of their immune microenvironment, including infiltrating cancer-associated fibroblasts (CAFs). C57BL/6J mice started treatment with RSV or DHS, solubilised in drinking water, one week before LLC implantation, and continued for 21 days, at the end of which they were sacrificed, and the tumour masses collected. Histology was performed according to standard procedures; angiogenesis, cell proliferation and death, autophagy, infiltrating-immune cells, macrophages and fibroblasts were assessed by immunodetection assays. Both stilbenic compounds were able to contrast the tumour growth by increasing apoptosis and autophagy in LLC tumour masses. Additionally, they contrasted the tumour-permissive microenvironment by limiting the infiltration of tumour-associated immune-cells and, more importantly, by counteracting CAF maturation. Therefore, both stilbenes could be employed to synergise with conventional oncotherapies to limit the contribution of stromal cells in tumour growth.

Primary ciliopathies are rare inherited disorders caused by structural or functional defects in the primary cilium, a subcellular organelle present on the surface of most cells. Primary ciliopathies show considerable clinical and genetic heterogeneity, with disruption of over 100 genes causing the variable involvement of several organs, including the central nervous system, kidneys, retina, skeleton and liver. Pathogenic variants in one and the same gene may associate with a wide range of ciliopathy phenotypes, supporting the hypothesis that the individual genetic background, with potential additional variants in other ciliary genes, may contribute to a mutational load eventually determining the phenotypic manifestations of each patient. Functional studies in animal models have uncovered some of the pathophysiological mechanisms linking ciliary gene mutations to the observed phenotypes; yet, the lack of reliable human cell models has previously limited preclinical research and the development of new therapeutic strategies for primary ciliopathies. Recent technical advances in the generation of patient-derived two-dimensional (2D) and three-dimensional (3D) cellular models give a new spur to this research, allowing the study of pathomechanisms while maintaining the complexity of the genetic background of each patient, and enabling the development of innovative treatments to target specific pathways. This review provides an overview of available models for primary ciliopathies, from existing in vivo models to more recent patient-derived 2D and 3D in vitro models. We highlight the advantages of each model in understanding the functional basis of primary ciliopathies and facilitating novel regenerative medicine, gene therapy and drug testing strategies for these disorders., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. No commercial re-use. See rights and permissions. Published by BMJ.)

An innovative approach was developed to engineer a multi-layered chitosan scaffold for osteochondral defect repair. A combination of freeze drying and porogen-leaching out methods produced a porous, bioresorbable scaffold with a distinct gradient of pore size (mean = 160-275 μm). Incorporation of 70 wt% nano-hydroxyapatite (nHA) provided additional strength to the bone-like layer. The scaffold showed instantaneous mechanical recovery under compressive loading and did not delaminate under tensile loading. The scaffold supported the attachment and proliferation of human mesenchymal stem cells (MSCs), with typical adherent cell morphology found on the bone layer compared to a rounded cell morphology on the chondrogenic layer. Osteogenic and chondrogenic differentiation of MSCs preferentially occurred in selected layers of the scaffold in vitro, driven by the distinct pore gradient and material composition. This scaffold is a suitable candidate for minimally invasive arthroscopic delivery in the clinic with potential to regenerate damaged cartilage and bone., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Adult neurogenesis is well-described in the subventricular and subgranular zones of the mammalian brain. Recent observations that resident glia express stem cell markers in some areas of the brain not traditionally associated with neurogenesis hint to a possible role in tissue repair. The Bergmann glia (BG) population in the cerebellum displays markers and in vitro features associated with neural stem cells (NSC), however the physiological relevance of this phenotypic overlap remains unclear in the absence of established in vivo evidence of tissue regeneration in the adult cerebellum. Here, this BG population was analysed in the adult cerebellum of different species and showed conservation of NSC-associated marker expression including Sox1, Sox2 and Sox9, in chick, primate and mouse cerebellum tissue. NSC-like cells isolated from adult mouse cerebellum showed slower growth when compared to lateral ventricle NSC, as well as differences upon differentiation. In a mouse model of cerebellar degeneration, progressive Purkinje cell loss was linked to cerebellar cortex disorganisation and a significant increase in Sox-positive cells compared to matching controls. These results show that this Sox-positive population responds to cerebellar tissue disruption, suggesting it may represent a mobilisable cellular resource for targeted strategies to promote tissue repair., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)