All roads lead to Rome: the many ways to pluripotency

Cell pluripotency, spatial restriction, and development are spatially and temporally controlled by epigenetic regulatory mechanisms that occur without any permanent loss or alteration of genetic material, but rather through modifications "on top of it." These changes modulate the accessibility to transcription factors, either allowing or repressing their activity, thus shaping cell phenotype. Several studies have demonstrated the possibility to interact with these processes, reactivating silenced genes and inducing a high plasticity state, via an active demethylating effect, driven by ten-eleven translocation (TET) enzymes and an overall decrease of global methylation. In agreement with this, TET activities have been shown to be indispensable for mesenchymal to epithelial transition of somatic cells into iPSCs and for small molecule-driven epigenetic erasure. Beside the epigenetic mechanisms, growing evidences highlight the importance of mechanical forces in supporting cell pluripotency, which is strongly influenced by 3D rearrangement and mechanical properties of the surrounding microenvironment, through the activation of specific mechanosensing-related pathways. In this review, we discuss and provide an overview of small molecule ability to modulate cell plasticity and define cell fate through the activation of direct demethylating effects. In addition, we describe the contribution of the Hippo signaling mechanotransduction pathway as one of the mechanisms involved in the maintenance of pluripotency during embryo development and its induction in somatic cells

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

Cellular and molecular mechanisms regulating oocyte quality and the relevance for farm animal reproductive efficiency

The efficiency of breeding schemes is dependent on the high fecundity of the selected individuals. Reproductive technologies are constantly pushing the physiological limits, but while the male reproductive potential is almost fully exploited, female reproductive physiology is the subject of constant research. Since the number of offspring that a female can bring to term each pregnancy cannot be changed, the ideal approach is to remove the potential offspring at the beginning of development and to transfer them to recipients of lesser genetic value. The earlier the collection takes place, the higher the number of descendants that a female can generate, so that now, the number of available oocytes becomes the limiting factor. This article will describe how detailed studies on oocyte physiology are beginning to unravel the complex sequence that transforms a small primordial follicle into a large ovulatory follicle containing a mature oocyte. Progressively, the limits to oocyte manipulation have been recognised and gradually overcome with adequate hormonal treatments in vivo and with specific media supplementation in vitro. This has led to the development of highly efficient reproductive technologies and the promise of even greater advances in the future. Surprising new findings, such as ovarian stem cells that can replenish the follicle population or long term embryonic stem cell lines that can differentiate into oocytes, are rapidly changing our expectations

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

Bridging the gap between cell culture and live tissue

Traditional in vitro two-dimensional (2-D) culture systems only partly imitate the physiological and biochemical features of cells in their original tissue. In vivo, in organs and tissues, cells are surrounded by a three-dimensional (3-D) organization of supporting matrix and neighbouring cells, and a gradient of chemical and mechanical signals. Furthermore, the presence of blood flow and mechanical movement provides a dynamic environment (Jong et al., 2011). In contrast, traditional in vitro culture, carried out on 2-D plastic or glass substrates, typically provides a static environment, which, however is the base of the present understanding of many biological processes, tissue homeostasis as well as disease. It is clear that this is not an exact representation of what is happening in vivo and the microenvironment provided by in vitro cell culture models are significantly different and can cause deviations in cell response and behaviour from those distinctive of in vivo tissues. In order to translate the present basic knowledge in cell control, cell repair and regeneration from the laboratory bench to the clinical application, we need a better understanding of the cell and tissue interactions. This implies a detailed comprehension of the natural tissue environment, with its organization and local signals, in order to more closely mimic what happens in vivo, developing more physiological models for efficient in vitro systems. In particular, it is imperative to understand the role of the environmental cues which can be mainly divided into those of a chemical and mechanical nature

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

The quest for an effective and safe personalized cell therapy using epigenetic tools

In the presence of different environmental cues that are able to trigger specific responses, a given genotype has the ability to originate a variety of different phenotypes. This property is defined as plasticity and allows cell fate definition and tissue specialization. Fundamental epigenetic mechanisms drive these modifications in gene expression and include DNA methylation, histone modifications, chromatin remodeling, and microRNAs. Understanding these mechanisms can provide powerful tools to switch cell phenotype and implement cell therapy. Environmentally influenced epigenetic changes have also been associated to many diseases such as cancer and neurodegenerative disorders, with patients that do not respond, or only poorly respond, to conventional therapy. It is clear that disorders based on an individual\u2019s personal genomic/epigenomic profile can rarely be successfully treated with standard therapies due to genetic heterogeneity and epigenetic alterations and a personalized medicine approach is far more appropriate to manage these patients. We here discuss the recent advances in small molecule approaches for personalized medicine, drug targeting, and generation of new cells for medical application. We also provide prospective views of the possibility to directly convert one cell type into another, in a safe and robust way, for cell-based clinical trials and regenerative medicine

A Detailed Study of Rainbow Trout (Onchorhynchus mykiss) Intestine Revealed That Digestive and Absorptive Functions Are Not Linearly Distributed along Its Length

To increase the sustainability of trout farming,the industry requires alternatives to \ufb01sh-based meals that do not compromise animal health and growth performances. To develop new feeds, detailed knowledge of intestinal morphology and physiology is required. We performed histological, histochemical, immunohistochemical and morphometric analysis at typical time points of in vivo feeding trials (50, 150 and 500 g). Only minor changes occurred during growth whereas di\ufb00erences characterized two compartments, not linearly distributed along the intestine. The \ufb01rst included the pyloric caeca, the basal part of the complex folds and the villi of the distal intestine. This was characterized by a signi\ufb01cantly smaller number of goblet cells with smaller mucus vacuoles, higher proliferation and higher apoptotic rate but a smaller extension of fully di\ufb00erentiated epithelial cells and by the presence of numerous pinocytotic vacuolization. The second compartment was formed by the proximal intestine and the apical part of the posterior intestine complex folds. Here we observed more abundant goblet cells with bigger vacuoles, low proliferation rate, few round apoptotic cells, a more extended area of fully di\ufb00erentiated cells and no pinocytotic vacuoles. Our results suggest that rainbow trout intestine is physiologically arranged to mingle digestive and absorptive functions along its lengt

in search of the transcriptional blueprints of a competent oocyte

The oocyte undergoes a remarkably long and elaborated journey within the follicle before becoming fully equipped to sustain embryonic development. Its ability to support early embryonic development relies largely on the maternal transcripts accumulated during its growth and maturation. However, it is still not clear what transcriptome blueprint composes a competent oocyte. A number of extensive studies provided a detailed characterization of the mRNA molecules that are gradually accumulated in the oocyte cytoplasm. The detail of our knowledge has gradually increased through the years also thanks to the development and improvement of the analytical techniques. From realtime PCR analysis of single transcripts, to the whole transcriptome approach of gene arrays and new genereation sequencing, scientists accumulated an exponentially growing amount of new information. More recently, the discovery of non-coding RNAs revealed a new layer of complexity in the mechanisms that modulate gene expression at the mRNA level, in folliculogenesis and oogenesis. In particular, data are emerging on the potential role of microRNAs in controlling ovarian function, oocyte maturation and the oocyte-somatic cell cross talk. This review will try to summarize the vast amount of data currently available on the mRNAs and microRNAs associated with the ovarian function and to find their biological significance