Extracellular vesicles (EVs) have prevalent roles in cancer biology and regenerative medicine. Conventional techniques for characterising EVs including electron microscopy (EM), nanoparticle tracking analysis (NTA) and tuneable resistive pulse sensing (TRPS), have been reported to produce high variability in particle count (EM) and poor sensitivity in detecting EVs below 50 nm in size (NTA and TRPS), making accurate and unbiased EV analysis technically challenging. This study introduces direct stochastic optical reconstruction microscopy (d-STORM) as an efficient and reliable characterisation approach for stem cell-derived EVs. Using a photo-switchable lipid dye, d-STORM imaging enabled rapid detection of EVs down to 20-30 nm in size with higher sensitivity and lower variability compared to EM, NTA and TRPS techniques. Imaging of EV uptake by live stem cells in culture further confirmed the potential of this approach for downstream cell biology applications and for the analysis of vesicle-based cell-cell communication.

Brown adipose tissue (BAT) is able to rapidly generate heat and metabolise macronutrients, such as glucose and lipids, through activation of mitochondrial uncoupling protein 1 (UCP1). Diet can modulate UCP1 function but the capacity of individual nutrients to promote the abundance and activity of UCP1 is not well established. Caffeine consumption has been associated with loss of body weight and increased energy expenditure, but whether it can activate UCP1 is unknown. This study examined the effect of caffeine on BAT thermogenesis in vitro and in vivo. Stem cell-derived adipocytes exposed to caffeine (1 mM) showed increased UCP1 protein abundance and cell metabolism with enhanced oxygen consumption and proton leak. These functional responses were associated with browning-like structural changes in mitochondrial and lipid droplet content. Caffeine also increased peroxisome proliferator-activated receptor gamma coactivator 1-alpha expression and mitochondrial biogenesis, together with a number of BAT selective and beige gene markers. In vivo, drinking coffee (but not water) stimulated the temperature of the supraclavicular region, which co-locates to the main region of BAT in adult humans, and is indicative of thermogenesis. Taken together, these results demonstrate that caffeine can promote BAT function at thermoneutrality and may have the potential to be used therapeutically in adult humans.

5-Methylcytosine (5mC) is an epigenetic modification involved in regulation of gene activity during differentiation. Tet dioxygenases oxidize 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). Both 5fC and 5caC can be excised from DNA by thymine-DNA glycosylase (TDG) followed by regeneration of unmodified cytosine via the base excision repair pathway. Despite evidence that this mechanism is operative in embryonic stem cells, the role of TDG-dependent demethylation in differentiation and development is currently unclear. Here, we demonstrate that widespread oxidation of 5hmC to 5caC occurs in postimplantation mouse embryos. We show that 5fC and 5caC are transiently accumulated during lineage specification of neural stem cells (NSCs) in culture and in vivo. Moreover, 5caC is enriched at the cell-type-specific promoters during differentiation of NSCs, and TDG knockdown leads to increased 5fC/5caC levels in differentiating NSCs. Our data suggest that active demethylation contributes to epigenetic reprogramming determining lineage specification in embryonic brain.

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.