Role of oxygen tension and genetic background during the epigenetic conversion of mouse fibroblasts into insulin secreting cells

Epigenetic cell conversion overcomes the stability of a mature cell phenotype transforming a somatic cell in an unlimited source of autologous cells of a different type. It is based on the exposure to a demethylating agent followed by an induction protocol. In our work we exposed mouse dermal fibroblasts to the demethylating agent 5-azacytidine. Cell differentiation was directed toward the endocrine pancreatic lineage with a sequential combination of Activin A, Retinoic Acid, B27 supplement, ITS and bFGF. The overall duration of the process was 10 days. Aim of this work was to evaluate the role of oxygen during differentiation of dermal fibroblasts derived from two different mouse strains, NOD and C57 BL/6J. During differentiation, both cell lines were cultured either in the standard in vitro culture 20% oxygen concentration or in the lower and more physiological 5% of oxygen. Our results show that C57 BL/6J cells are able to differentiate into insulin secreting cells in both oxygen tensions with a higher amount of insulin release in low oxygen conditions. On the other hand, cells of NOD mice, which are physiologically predisposed to the onset of diabetes, differentiate in 20% of oxygen but not in low oxygen and they died after three days of culture. However, if these cells are moved to 5% of oxygen after their differentiation in high oxygen they remain viable for up to four days. Furthermore, their capacity to release insulin remains unchanged for 24 hours. Results suggest that genetic background has a profound effect on the role of oxygen during the in vitro differentiation process, possibly reflecting the different susceptibility to the disease of the strains used in the experiment.Supported by EFSD and Carraresi Foundatio

High glucose concentrations are required for endocrine pancreatic differentiation of mammalian adult fibroblasts.

Epigenetic conversion overcomes the stability of a terminally differentiated cell, allowing phenotypeswitch and providing an unlimited source of autologous cells of a different type. It is based on theexposure to an epigenetic modifier that increases cell plasticity, followed by a differentiation protocol.In our work we treat mammalian dermal fibroblasts with the demethylating agent 5-azacytidine. Celldifferentiation is directed toward the endocrine pancreatic lineage, with a sequential combination ofkey growth factors. The overall duration of the process is 36 days (Pennarossa, 2013; Brevini, 2015; Brevini,2015). However, this protocol, as well as all differentiation procedures described in the literature, useshigh and non-physiological concentrations of glucose. Here we report experiments aimed atinvestigating whether the use of lower glucose concentrations, that more closely mimic the in vivophysiological environment, can support fibroblast conversion into β-like cells. To do so, cells werecultured as described above, but using lower and more physiological glucose levels, namely 5.5 and 8.5mM that correspond to normoglycaemia before and after meals (International Diabetes Federation,2007). Our results show that mammalian cells are not able to differentiate into insulin secreting cells ina low glucose environment. In particular, cells do not aggregate into pancreatic islet structures anddisplay an altered gene expression pattern for several early pancreatic markers, when compared to thestandard trend obtained with 17.5 mM of glucose. These results suggest that high glucose levels areessential for the achievement of the endocrine pancreatic differentiation process in mammalian cellsand appear to be crucial for functional efficiency and morphological organization

Liquid Marble micro-bioreactor promotes 3D cell rearrangement and induces, maintains and stabilizes high plasticity in epigenetically erased fibroblasts

In the last years, many works demonstrated the possibility to directly interact with the epigenetic signature of an adult mature cell, through the use of epigenetic modifiers, (Pennarossa et al., 2013; Brevini et al., 2014, Chandrakantan et al., 2016) and new mechanisms underlying this process have been recently described (Manzoni et al., 2016). In particular, the small molecule 5-azacytidine (5-aza-CR) has been shown to induce a transient higher plasticity state in adult somatic cells, grown in standard 2D conditions. Recent evidence have also shown the possibility to regulate and maintain cell pluripotency through the use of 3D culture systems. In the experiments here presented, we combine the two approaches and investigate whether the simultaneous use of a 3D micro-bioreactor and 5-aza-CR is able to promote cell rearrangement, boost the induction of high plasticity and stably maintain it.To this purpose, fibroblasts were either plated on plastic dishes (2D) or encapsulated in a Liquid Marble (LM) micro-bioreactor (polytetrafluoroethylene (PTFE)), which has been previously shown to support the growth of living microorganisms, tumor spheroids, fibroblasts, red blood cells, and embryonic stem cells (Ledda et al., 2016). Cells were then erased with 5-aza-CR, for 18 hours and cultured in Embryonic Stem Cell (ESC) medium for up to 28 days. Morphological analysis and pluripotency related gene expression levels were monitored for the entire length of the experiments. 2D cells, kept a monolayer pattern and acquired a pluripotent state that was, however, transient and lost by day 6. In contrast the use of a 3D system maintained and stabilized the high plasticity state in LM cells until the end of the experiments (Fig. 1). The data obtained demonstrate that cell rearrangement and interactions may modulate 5-aza-CR induced plasticity and suggest a correlation between 3D mechano-transduction-related pathways and  epigenetic regulation of cell phenotype

Matrix stiffness boosts pancreatic differentiation via the YAP/TAZ mechanotransduction mediated pathway

In the last years, many papers highlighted the possibility to use epigenetic modifiers to directly interact with the epigenetic signature of an adult mature cell (Pennarossa et al., 2013; Chandrakantan et al., 2016). In particular, the molecule 5-azacytidine (5-aza-CR), which is able to interfere with DNA methylation, through both a direct and an indirect effect (Manzoni et al., 2016), can be used to remove the epigenetic ‘blocks’ responsible for tissue specification and to facilitate  cell transition to a different lineage. In parallel, recent evidence has also shown that epigenetic conversion is influenced by the 3D rearrangement and by the mechanical properties of the cellular microenvironment (Pennarossa et al., 2017). In the experiments here presented, we investigated the effect of a selected 3D culture system on the conversion process. We used INS-eGFP porcine fibroblasts, that express enhanced green fluorescent protein (eGFP) under the control of insulin gene promoter, as experimental model, and wild-type pig fibroblasts, as control. Both cell types, were plated either on plastic or on 1kPa polyacrylamide (PAA) gel, that mimics the stiffness of pancreatic tissue in vivo. Cells were erased with 5-aza-CR for 18h and exposed to specific differentiation stimuli for 36 days (Pennarossa et al., 2014). The use of INS-eGFP fibroblasts allowed real-time monitoring of cells progressing towards the pancreatic phenotype. Morphological analysis and pancreatic marker expression were checked for the entire length of the experiment. PAA gels encouraged the induction of islet-like structures, suggesting that the of tridimensional clusters may be a crucial aspect of pancreatic differentiation in vitro. Moreover, the use of an adequate substrate accelerated cell differentiation process and anticipated insulin secretion ability. The results obtained demonstrated the direct implication of the yes-associated protein/transcriptional co-activator with PDZ-binding motif (YAP/TAZ) mechanotransduction-mediated pathway, indicating  that mechanical cues exert a key role in pancreatic phenotype definition

CCD and photon-counting photometric observations of asteroids carried out at Padova and Catania observatories

We present the results of observational campaigns of asteroids performed at Asiago Station of Padova Astronomical Observatory and at M.G. Fracastoro Station of Catania Astrophysical Observatory, as part of the large research programme on Solar System minor bodies undertaken since 1979 at the Physics and Astronomy Department of Catania University. Photometric observations of six Main-Belt asteroids (27 Euterpe, 173 Ino, 182 Elsa, 539 Pamina, 849 Ara, and 984 Gretia), one Hungaria (1727 Mette), and two Near-Earth Objects (3199 Nefertiti and 2004 UE) are reported. The first determination of the synodic rotational period of 2004 UE was obtained. For 182 Elsa and 1727 Mette the derived synodic period of 80.23+/-0.08 h and 2.981+/-0.001 h, respectively, represents a significant improvement on the previously published values. For 182 Elsa the first determination of the H-G magnitude relation is also presented.Comment: 19 pages, 11 figures, accepted for publication in Planetary and Space Scienc

Pluripotency in Domestic Animal Cells

VII, 46 p. 28 illus., 24 illus. in color.online r

Matrix stiffness and oxigen tension modulate epigenetic conversion of mouse dermal fibroblasts into insulin producing cells.

In vivo, cells are surrounded by a three-dimensional (3-D) organization of supporting matrix, neighboring cells and a gradient of chemical and mechanical signals (Antoni, et al., 2015). However, the present understanding of many biological processes is mainly based on two-dimensional (2-D) systems that typically provides a static environment. In the present study, we tested two different 3-D culture systems and apply them to the epigenetic conversion of mouse dermal fibroblasts into insulin producing-cells (Pennarossa, et al., 2013; Brevini, et al., 2015), combining also the use of two oxygen tensions. In particular, cells were differentiated using the Polytetrafluoroethylene micro-bioreactor (PTFE) and the Polyacrylamide (PAA) gels with different stiffness (1 kPa; 4 kPa), maintained either in the standard 20% or in the more physiological 5% oxygen tensions. Standard differentiation performed on plastic substrates was assessed as a control. Cell morphology (Fig.1A), insulin expression and release were analyzed to evaluate the role of both stiffness and oxygen tension in the process. The results obtained showed that 1 kPa PAA gel and PTFE system induced a significantly higher insulin expression and release than plastic and 4 kPa PAA gel, especially in low oxygen condition (Fig.1B). Furthermore, comparing the efficiency of the two systems tested, 1 kPa PAA gel ensured a higher insulin transcription than PTFE (Fig.1C). Recent studies show the direct influence of substrates on lineage commitment and cell differentiation (Engler, et al., 2006; Evans, et al., 2009). The evidence here presented confirm that the use of an appropriate stiffness (similar to the pancreatic tissue), combined with a physiological oxygen tension, promote β-cell differentiation, with beneficial effects on cell functional activity and insulin release. The present results highlight the importance of 3-D cell rearrangement and oxigen tension to promote in vitro epigenetic conversion of mouse fibroblasts into insulin-producing cells.