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

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

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

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

Implications of miRNA expression pattern in bovine oocytes and follicular fluids for developmental competence

Developmental competence determines the oocyte capacity to support initial embryo growth, but the molecular mechanisms underlying this phenomenon are still ill-defined. Changes in microRNA (miRNA) expression pattern have been described during follicular growth in several species. Therefore, aim of this study was to investigate whether miRNA expression pattern in cow oocyte and follicular fluid (FF) is associated with the acquisition of developmental competence. Samples were collected from ovaries with more than, or fewer than, 10 mid-antral follicles (H- and L-ovaries) because previous studies demonstrated that this parameter is a reliable predictor of oocyte competence. After miRNA deep sequencing and bioinformatic data analysis, we identified 58 miRNAs in FF and 6 in the oocyte that were differentially expressed between H- and L-ovaries. Overall, our results indicate that miRNA levels both in FF and in the ooplasm must remain within specific thresholds and that changes in either direction compromising oocyte competence. Some of the miRNAs found in FF (miR-769, miR-1343, miR-450a, miR-204, miR-1271 and miR-451) where already known to regulate follicle growth and their expression pattern indicate that they are also involved in the acquisition of developmental competence. Some miRNAs were differentially expressed in both compartments but with opposite patterns, suggesting that miRNAs do not flow freely between FF and oocyte. Gene Ontology analysis showed that the predicted gene targets of most differentially expressed miRNAs are part of a few signalling pathways. Regulation of maternal mRNA storage and mitochondrial activity seem to be the processes more functionally relevant in determining oocyte quality. In conclusion, our data identified a few miRNAs in the follicular fluid and in the ooplasm that modulate the oocyte developmental competence. This provides new insights that could help with the management of cattle reproductive efficiency

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

Mountain high and valley deep: epigenetic controls of pluripotency and cell fate

All the somatic cells composing a mammalian organism are genetically identical and contain the same DNA sequence. Nevertheless, they are able to adopt a distinct commitment, differentiate in a tissue specific way and respond to developmental cues, acquiring a terminal phenotype. At the end of the differentiation process, each cell is highly specialized and committed to a distinct determined fate. This is possible thanks to tissue-specific gene expression, timely regulated by epigenetic modifications, that gradually limit cell potency to a more restricted phenotype-related expression pattern. Complex chemical modifications of DNA, RNA and associated proteins, that determine activation or silencing of certain genes are responsible for the 'epigenetic control' that triggers the restriction of cell pluripotency, with the acquisition of the phenotypic definition and the preservation of its stability during subsequent cell divisions. The process is however reversible and may be modified by biochemical and biological manipulation, leading to the reactivation of hypermethylated pluripotency genes and inducing cells to transit from a terminally committed state to a higher plasticity one. These epigenetic regulatory mechanisms play a key role in embryonic development since they drive phenotype definition and tissue differentiation. At the same time, they are crucial for a better understanding of pluripotency regulation and restriction, stem cell biology and tissue repair process

Large animal models for cardiac stem cell therapies

Cardiovascular disease is the leading cause of death in developed countries and is one of the leading causes of disease burden in developing countries. Therapies have markedly increased survival in several categories of patients, nonetheless mortality still remains high. For this reason high hopes are associated with recent developments in stem cell biology and regenerative medicine that promise to replace damaged or lost cardiac muscle with healthy tissue, and thus to dramatically improve the quality of life and survival in patients with various cardiomyopathies. Much of our insight into the molecular and cellular basis of cardiovascular biology comes from small animal models, particularly mice. However, signi\ufb01cant differences exist with regard to several cardiac characteristics when mice are compared with humans. For this reason, large animal models like dog, sheep and pig have a well established role in cardiac research. A distinct characteristic of cardiac stem cells is that they can either be endogenous or derive from outside the heart itself; they can originate as the natural course of their differentiation programme (e.g., embryonic stem cells) or can be the result of speci\ufb01c inductive conditions (e.g., mesenchymal stem cells). In this review we will summarize the current knowledge on the kind of heart-related stem cells currently available in large animal species and their relevance to human studies as pre-clinical models

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