Tiziana Angela Luisa Brevini

Parthenogenetic Activation: Biology and Applications in the ART Laboratory

Parthenogenesis is a reproductive strategy typical of lower species where a female gives birth to offsprings without a paternal contribution. On the contrary, parthenogenesis is not a form of natural reproduction in mammals even if mammalian oocytes, under appropriate stimuli, can undergo to parthenogenetic activation. This review describes the biological mechanisms regulating parthenogenetic activation in mammals and illustrates the fundamental differences between embryos and parthenotes. Ethical, legal and political concerns on the value of human embryos regulate and limit human embryological studies founded on the widespread belief that human embryos should not be created and studied for research purposes only. Based on the differences between parthenotes and embryos the use of parthenogenesis is proposed as an experimental tool to investigate embryo development which may solve many of the ethical concerns associated with the use of human embryos for experimental purposes. Examples of the possible uses of parthenotes in many field of research such as in vitro assays aimed to study some aspects of assisted reproductive technologies (ART), toxicology or stem cell are described and their validity is discussed.

Development, embryonic genome activity and mitochondrial characteristics of bovine–pig inter-family nuclear transfer embryos

The best results of inter-species somatic cell nuclear transfer (iSCNT) in mammals were obtained using closely related species that can hybridise naturally. However, in the last years, many reports describing blastocyst development following iSCNT between species with distant taxonomical relations (inter-classes, inter-order and inter-family) have been published. This indicates that embryonic genome activation (EGA) in xeno-cytoplasm is possible, albeit very rarely. Using a bovine-pig (inter-family) iSCNT model, we studied the basic characteristics of EGA: expression and activity of RNA polymerase II (RNA Pol II), formation of nucleoli (as an indicator of RNA polymerase I (RNA Pol I) activity), expression of the key pluripotency gene NANOG and alteration of mitochondrial mass. In control embryos (obtained by IVF or iSCNT), EGA was characterised by RNA Pol II accumulation and massive production of poly-adenylated transcripts (detected with oligo dT probes) in blastomere nuclei, and formation of nucleoli as a result of RNA Pol I activity. Conversely, iSCNT embryos were characterised by the absence of accumulation and low activity of RNA Pol II and inability to form active mature nucleoli. Moreover, in iSCNT embryos, NANOG was not expressed, and mitochondria mass was significantly lower than in intra-species embryos. Finally, the complete developmental block at the 16-25-cell stage for pig-bovine iSCNT embryos and at the four-cell stage for bovine-pig iSCNT embryos strongly suggests that EGA is not taking place in iSCNT embryos. Thus, our experiments clearly demonstrate poor nucleus-cytoplasm compatibility between these animal species.

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.

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).xa0 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 xa0epigenetic regulation of cell phenotype.

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

Epigenetic conversion overcomes the stability of a terminally differentiated cell, allowing phenotype switch and providing an unlimited source of autologous cells of a different type. It is based on the exposure 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. Cell differentiation is directed toward the endocrine pancreatic lineage, with a sequential combination of key 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, uses high and non-physiological concentrations of glucose. Here we report experiments aimed at investigating whether the use of lower glucose concentrations, that more closely mimic the in vivo physiological environment, can support fibroblast conversion into β-like cells. To do so, cells were cultured as described above, but using lower and more physiological glucose levels, namely 5.5 and 8.5 mM 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 in a low glucose environment. In particular, cells do not aggregate into pancreatic islet structures and display an altered gene expression pattern for several early pancreatic markers, when compared to the standard trend obtained with 17.5 mM of glucose. These results suggest that high glucose levels are essential for the achievement of the endocrine pancreatic differentiation process in mammalian cells and appear to be crucial for functional efficiency and morphological organization.

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’s 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.