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

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

Use of a micro-bioreactor to promote 3-dimensional cell rearrangement and induce, maintain, and stabilize high plasticity in epigenetically erased fibroblasts

Development and cell differentiation are driven by complex epigenetic mechanisms that regulate chromatin structure and specific gene transcription programs. We recently demonstrated that it is possible to modify the epigenetic signature of terminally differentiated cells, switching their phenotype into one of higher plasticity, through the use of molecules that remove epigenetic marks from DNA and histones (Pennarossa et al. 2013 Proc. Natl. Acad. Sci. 110, 8948-8953; Brevini et al. 2014 Stem Cell Rev. 10, 633-642). Here we drive mammalian fibroblasts into a high plasticity state using the epigenetic eraser, 5-aza-cytidine (5-aza-CR), and investigate whether the simultaneous use of a micro-bioreactor culture system is able to promote three-dimensional (3D) cell rearrangement, boost the induction of high plasticity, and stably maintain it. To this purpose, fibroblasts were either plated on plastic dishes (Group A) or encapsulated in a liquid marble micro-bioreactor (polytetrafluoroethylene powder; Sigma 430935, St. Louis, MO; Group B). Both groups were erased with 5-aza-CR and cultured in embryonic stem cell medium for 28 days. Morphological analysis was carried out for the entire length of the experiment. The OCT4, NANOG, and REX1 expression levels were assessed by real-time PCR at different time points. Exposure to 5-aza-CR induced a dramatic change in morphology in Group A fibroblasts. Cells became rounded, with larger and granulated nuclei and retained a monolayer distribution for the entire length of the experiment. The same changes in cell and nuclear morphology were observed also in cells encapsulated in liquid marble (Group B). In addition, these cells formed 3D spherical structures that were stably maintained until Day 28. These morphological rearrangements were accompanied by the active expression of the pluripotency markers, OCT4, NANOG, and REX1, in both groups. However, while Group A cells progressively down-regulated their expression by Day 6, Group B cells steadily transcribed these genes until Day 28, when cultures were arrested. Altogether, the data confirm that epigenetic erasing induces a high plasticity state in terminally differentiated fibroblasts with the expression of pluripotency related genes. Striking morphological changes accompanied the removal of epigenetic marks. These were influenced by the use of an adequate 3D in vitro culture system, with the induction of distinctive cell rearrangements and the formation of spherical structures that boosted and maintained cell plasticity. These results suggest a correlation between the mechanotransduction pathways induced by the micro-bioreactor culture system and the epigenetic regulation of cell phenotype

5-azacytidine affects TET2 and histone transcription and reshapes morphology of human skin fibroblasts

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

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

MOLECULAR AND CELLULAR MECHANISMS REGULATING EPIGENETIC CELL CONVERSION

Phenotype definition is controlled by epigenetic regulations that allow cells to acquire their differentiated state. The process is reversible and cells can be driven back to a higher plasticity state with several different approaches, which include the interaction with the epigenetic set up through the use of epigenetic erasers, readers or writers. Among the many epigenetic modifiers presently available, in our previous experiments, we selected 5-azacytidine (5-aza-CR), which is a well-known DNA methyltransferase inhibitor, and has been previously shown to increase cell plasticity and facilitate phenotype changes in different cell types. Beside the epigenetic mechanisms driving cell conversion processes, growing evidences highlight the importance of mechanical forces that directly influence cell plasticity and differentiation. Aims of my PhD were: a) characterization of the molecular and cellular mechanisms regulating cell phenotype b) analysis of the possible relation between mechano-sensing and epigenetic control of cell plasticity and differentiation. The experiments carried out confirmed the global demethylating effect of 5-aza-CR, with a transient upregulation of pluripotency markers. At the same time increased transcription of TET2 and several histones was detected. This was accompanied by changes in enzymes controlling histone acetylation and cell morphological rearrangement. Interestingly, the study of DNA methylation profile and its regulatory genes, revealed that the use of a 3D micro-bioreactor promotes and stabilizes the maintenance of the acquired plasticity for a long period of culture. In particular, the use of an adequate soft substrate increased pancreatic conversion efficiency and induced the acquisition of a mono-hormonal phenotype, which is distinctive of terminally matured cell. Altogether these findings indicate that 5-aza-CR induced somatic cell transition to a higher plasticity state may be the result of multiple regulatory mechanisms that accompany the demethylating effect exerted by the modifier. The results described in my thesis also revealed that mechano-transduction-related responses modulate and maintain 5-aza- CR induced cell plasticity and significantly improve cell differentiation toward the pancreatic lineage

Methylation mechanisms and biomechanical effectors controlling cell fate

Mammalian development and cell fate specification are controlled by multiple regulatory mechanisms that interact in a coordinated way to ensure proper regulation of gene expression and spatial restriction, allowing cells to adopt distinct differentiation traits and a terminal phenotype. For example, cell potency is modulated by changes in methylation that are under the control of methyltransferases and ten-eleven translocation (TET) enzymes, which establish or erase a phenotype-specific methylation pattern during embryo development and mesenchymal to epithelial transition (MET). Cell plasticity is also responsive to extracellular factors, such as small molecules that interact with cell fate definition and induce a transient pluripotent state that allows the direct conversion of an adult mature cell into another differentiated cell type. In addition, cell-secreted vesicles emerge as powerful effectors, capable of modifying cell function and phenotype and delivering different signals, such as octamer-binding transcription factor-4 (Oct4) and SRY (sex determining region Y)-box 2 (Sox2) mRNAs (implicated in the preservation of pluripotency), thus triggering epigenetic changes in the recipient cells. In parallel, mechanical properties of the cellular microenvironment and three-dimensional rearrangement can affect both cell potency and differentiation through marked effects on cytoskeletal remodelling and with the involvement of specific mechanosensing-related pathways

Adding a dimension to cell fate

Cell fate specification, gene expression and spatial restriction are process finely tuned by epigenetic regulatory mechanisms. At the same time, mechanical forces have been shown to be crucial to drive cell plasticity and boost differentiation. Indeed, several studies have demonstrated that transitions along different specification states are strongly influenced by 3D rearrangement and mechanical properties of the surrounding microenvironment, that can modulate both cell potency and differentiation, through the activation of specific mechanosensing-related pathways. An overview of small molecule ability to modulate cell plasticity and define cell fate is here presented and results, showing the possibility to erase the epigenetic signature of adult dermal fibroblasts and convert them into insulin-producing cells (EpiCC) are described. The beneficial effects exerted on such processes, when cells are homed on an adequate substrate, that shows \u201cin vivo\u201c tissue-like stiffness are also discussed and the contribution of the Hippo signalling mechano-transduction pathway as one of the mechanisms involved is examined. In addition, results obtained using a genetically modified fibroblast cell line, expressing the enhanced green fluorescent protein (eGFP) under the control of the porcine insulin gene (INS) promoter (INS-eGFP transgenic pigs), are reported. This model offers the advantage to monitor the progression of cell conversion in real time mode. All these observations have a main role in order to allow a swift scale-up culture procedure, essential for cell therapy and tissue engineering applied to human regenerative medicine, and fundamental to ensure an efficient translation process from the results obtained at the laboratory bench to the patient bedside. Moreover, the creation of reliable in vitro model represents a key point to ensure the development of more physiological models that, in turn, may reduce the number of animals used, implementing non-invasive investigations and animal welfare and protection

Erase and Rewind : Epigenetic Conversion of Cell Fate

The potential of cell therapy in regenerative medicine has greatly expanded thanks to the availability of sources of pluripotent cells. In particular, induced pluripotent stem cells (iPS) have dominated the scenario in the last years for their ability to proliferate and differentiate into specific cell types. Nevertheless, the concerns inherent to the cell reprogramming process, limit iPS use in therapy and pose questions on the long-term behavior of these cells. In particular, despite the development of virus-free methods for their obtainment, a major and persisting drawback, is related to the acquisition of a stable pluripotent state, that is un-physiological and may lead to cell instability. The increased understanding of epigenetic mechanisms has paved the way to the use of \u201csmall molecules\u201d and \u201cepigenetic modifiers\u201d that allow the fine tuning of cell genotype and phenotype. In particular, it was demonstrated that an adult mature cell could be directly converted into a different cell type with the use of these chemicals, obtaining a new patient-specific cell, suitable for cell therapy. This approach is simple and direct and may represent a very promising tool for the regenerative medicine of several and diverse degenerative diseases

Epigenetic Erasing and Pancreatic Differentiation of Dermal Fibroblasts into Insulin-Producing Cells are Boosted by the Use of Low-Stiffness Substrate

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