Phenotype definition is controlled by epigenetic regulations that allow cells to acquire their differentiated state. The process is reversible and attractive for therapeutic intervention and for the reactivation of hypermethylated pluripotency genes that facilitate transition to a higher plasticity state. We report the results obtained in human fibroblasts exposed to the epigenetic modifier 5-azacytidine (5-aza-CR), which increases adult cell plasticity and facilitates phenotype change. Although many aspects controlling its demethylating action have been widely investigated, the mechanisms underlying 5-aza-CR effects on cell plasticity are still poorly understood. Our experiments confirm decreased global methylation, but also demonstrate an increase of both Formylcytosine (5fC) and 5-Carboxylcytosine (5caC), indicating 5-aza-CR ability to activate a direct and active demethylating effect, possibly mediated via TET2 protein increased transcription. This was accompanied by transient upregulation of pluripotency markers and incremented histone expression, paralleled by changes in histone acetylating enzymes. Furthermore, adult fibroblasts reshaped into undifferentiated progenitor-like phenotype, with a sparse and open chromatin structure. Our findings indicate that 5-aza-CR induced somatic cell transition to a higher plasticity state is activated by multiple regulations that accompany the demethylating effect exerted by the modifier.

Several studies have demonstrated the possibility to revert differentiation process, reactivating hypermethylated genes and facilitating cell transition to a different lineage. Beside the epigenetic mechanisms driving cell conversion processes, growing evidences highlight the importance of mechanical forces in supporting cell plasticity and boosting differentiation. Here, we describe epigenetic erasing and conversion of dermal fibroblasts into insulin-producing cells (EpiCC), and demonstrate that the use of a low-stiffness substrate positively influences these processes. Our results show a higher expression of pluripotency genes and a significant bigger decrease of DNA methylation levels in 5-azacytidine (5-aza-CR) treated cells plated on soft matrix, compared to those cultured on plastic dishes. Furthermore, the use of low-stiffness also induces a significant increased up-regulation of ten-eleven translocation 2 (Tet2) and histone acetyltransferase 1 (Hat1) genes, and more decreased histone deacetylase enzyme1 (Hdac1) transcription levels. The soft substrate also encourages morphological changes, actin cytoskeleton re-organization, and the activation of the Hippo signaling pathway, leading to yes-associated protein (YAP) phosphorylation and its cytoplasmic translocation. Altogether, this results in increased epigenetic conversion efficiency and in EpiCC acquisition of a mono-hormonal phenotype. Our findings indicate that mechano-transduction related responsed influence cell plasticity induced by 5-aza-CR and improve fibroblast differentiation toward the pancreatic lineage.

24Developmental competence determines the oocyte capacity to support initial embryo 25 growth, but the molecular mechanisms underlying this phenomenon are still ill-26 defined. Changes in microRNA (miRNA) expression pattern have been described 27 during follicular growth in several species. Therefore, aim of this study was to 28 investigate whether miRNA expression pattern in cow oocyte and follicular fluid (FF) 29 is associated with the acquisition of developmental competence. Samples were 30 collected from ovaries with more than, or fewer than, 10 mid-antral follicles (H-and 31 L-ovaries) because previous studies demonstrated that this parameter is a reliable 32 predictor of oocyte competence. After miRNA deep sequencing and bioinformatic 33 data analysis, we identified 58 miRNAs in FF and 6 in the oocyte that were 34 differentially expressed between H-and L-ovaries. Overall, our results indicate that 35 miRNA levels both in FF and in the ooplasm must remain within specific thresholds 36 and that changes in either direction compromise oocyte competence. Some of the 37 miRNAs found in FF (miR-769, miR-1343, miR-450a, miR-204, miR-1271 and miR-38 451) where already known to regulate follicle growth and their expression pattern 39 indicate that they are also involved in the acquisition of developmental competence. 40Some miRNAs were differentially expressed in both compartments but with opposite 41 patterns, suggesting that miRNAs do not flow freely between FF and oocyte. Gene 42Ontology analysis showed that the predicted gene targets of most differentially 43 expressed miRNAs are part of a few signalling pathways. Regulation of maternal 44 mRNA storage and mitochondrial activity seem to be the processes more 45 functionally relevant in determining oocyte quality. In conclusion, our data identified a 46 few miRNAs in the follicular fluid and in the ooplasm that modulate the oocyte 47Recently, microRNAs (miRNAs), which regulate gene expression at the mRNA level, 65 have been associated with folliculogenesis and oogenesis [10, 11]. MiRNAs, which 66 range in size from 18 to 25 nucleotides (nt), have been found in the different 67 compartments of ovarian follicles, including granulosa cells [12, 13], theca cells [14], 68 follicular fluid and the oocyte itself [15]. Studies on the role of miRNAs during follicle 69 development in humans [16][17][18], mice [19, 20], cattle [10, 21, 22], pigs [23] and 70horses [24] suggest that they regulate the cellular differentiation processes which 71 occur during follicular development. 72 Follicular fluid and germinal vesicle oocyte collection 111Ovaries were collected at a commercial abattoir and were transported to the 112 laboratory in warmed (27-30°C) Dulbecco Phosphate Buffered Saline (PBS). Ovaries 113 were classified into low and high antral follicle count categories according to the 114 methods used in previous works [28, 29]. Briefly, the ovaries were assigned to high 115 antral follicle count ovaries (H ovaries) when more than 10 mid-antral follicles (2-5 116 mm in dia...

Phenotype definition is driven by epigenetic mechanisms as well as directly influenced by the cell microenvironment and by biophysical signals deriving from the extracellular matrix. The possibility to interact with the epigenetic signature of an adult mature cell, reversing its differentiated state and inducing a short transient high plasticity window, was previously demonstrated. In parallel, in vitro studies have shown that 3D culture systems, mimicking cell native tissue, exert significant effects on cell behavior and functions. Here we report the production of "PTFE micro-bioreactors" for long-term culture of epigenetically derived high plasticity cells. The system promotes 3D cell rearrangement, global DNA demethylation and elevated transcription of pluripotency markers, that is dependent on WW domain containing transcription regulator 1 (TAZ) nuclear accumulation and SMAD family member 2 (SMAD2) co-shuttling. Our findings demonstrate that the use of 3D culture strategies greatly improves the induction and maintenance of a high plasticity state.

Cell remarkable ability to self-organize and rearrange in functional organoids has been greatly boosted by the recent advances in 3-D culture technologies and materials. This approach can be presently applied to model human organ development and function "in a dish" and to predict drug response in a patient specific fashion.Here we describe a protocol that allows for the derivation of functional pancreatic mini-organoids from skin biopsies. Cells are suspended in a drop of medium and encapsulated with hydrophobic polytetrafluoroethylene (PTFE) powder particles, to form microbioreactors defined as "Liquid Marbles," that stimulate cell coalescence and 3-D aggregation. The PTFE shell ensures an optimal gas exchange between the interior liquid and the surrounding environment. It also makes it possible to scale down experiments and work in smaller volumes and is therefore amenable for higher throughput applications.