108 research outputs found
CENTROSOME BIOGENESIS AND ADAPTIVE RESPONSE IN MAMMAL PARTHENOGENETIC CELLS
No full textHuman parthenotes have been proposed as a source of embryonic stem cells despite the high incidence of aneuploidy described in parthenotes of most mammalian species. Through a comparative analysis between parthenogenetic and bi-parental cells lines we found that parthenogenetic cells are affected by chromosomal instability and centrosome amplification. We provide evidence that both alterations are determined by the lack of paternal centriole, normally contributed by the sperm at the time of fertilization, but parthenogenetic cell lines activate a series of adaptive mechanisms that allow them to proliferate and differentiate. These include down-regulation of the p53/p21 pathway, massive increase of autophagic activity and formation of a wide network of intercellular bridges with the morphological and molecular characters of blocked cell abscissions. These processes are commonly observed in transformed cells therefore parthenogenesis may be used to explore the mechanisms regulating oncogenesis and their link with self-renewal and pluripotency in human cell lines
All roads lead to Rome: the many ways to pluripotency
No full textCell 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
Beneficial effect of directional freezing on in vitro viability of cryopreserved sheep whole ovaries and ovarian cortical slices
No full textSTUDY QUESTION: Does directional freezing improve the structural and functional integrity of ovarian fragments compared with conventional slow freezing and to whole ovary cryopreservation? SUMMARY ANSWER: Compared with slow freezing, the use of directional freezing significantly improves all structural and functional parameters of ovarian fragments assessed in vitro and, overall, whole ovaries were better preserved than ovarian fragments. WHAT IS KNOWN ALREADY: Directional freezing has been developed to provide an alternative way to cryopreserve large biological samples and it is known to improve the structural and functional integrity of whole ovaries. Conventional slow freezing of ovarian fragments is the procedure more widely used in clinical settings but it causes substantial structural damage that limits the functional period after transfer back into the patient. STUDY DESIGN, SIZE, DURATION: We performed a 2 × 2 factorial design experiment on a total of 40 sheep ovaries, divided into four groups (n = 10 ovaries per group): (i) directional freezing of whole ovary (DFwo); (ii) directional freezing of ovarian fragments (DFof); (iii) conventional freezing of whole ovary (CFwo); (iv) conventional freezing of ovarian fragments (CFof). An additional eight ovaries were used as fresh controls. PARTICIPANTS/ MATERIALS, SETTING, METHODS: Ewe ovaries were randomly assigned to one of the experimental groups and frozen accordingly. Upon thawing, ovarian tissue was examined morphologically and cultured in vitro for 7 days. Samples were analyzed for cell proliferation and apoptosis, for DNA damage and repair activity, and for the presence of a panel of heat shock proteins (HSPs) by immunohistochemistry. MAIN RESULTS AND THE ROLE OF CHANCE: Most studied parameters were significantly improved (P < 0.05) in all samples cryopreserved with directional compared with slow freezing. The proportion of primordial follicles, which developed to the primary stage in whole ovaries (53 ± 1.7%) and in ovarian fragments (44 ± 1.8%) cryopreserved with directional freezing, was greater than with slow frozen whole ovaries (6 ± 0.5%, P = 0.001) or fragments (32 ± 1.5%, P = 0.004). After 7 days of culture, cell proliferation in DFwo (28 ± 0.73%) was the highest of all groups (P < 0.05) followed by DFof (23 ± 0.81%), CFof (20 ± 0.79%) and CFwo (9 ± 0.85%). Directional freezing also resulted in a better preservation of the cell capacity to repair DNA damage compared with slow freezing both in whole ovaries and ovarian fragments. Apoptosis and HSP protein levels were significantly increased only in the CFwo group. Direct comparison demonstrated that, overall, DFwo had better parameters than DFof and was no different from the fresh controls. LIMITATIONS, REASONS FOR CAUTION: The study is limited to an in vitro evaluation and uses sheep ovaries, which are smaller than human ovaries and therefore may withstand the procedures better. WIDER IMPLICATIONS OF THE FINDINGS: Improved integrity of ovarian morphology may translate to improved outcomes after transplantation. Alternatively, the particularly good preservation of whole ovaries suggests they could provide a source of ovarian follicles for in vitro culture in those cases when the presence of malignant cells poses a substantial risk for the patient. STUDY FUNDING/COMPETING INTEREST(S): Supported by: Associazione Italiana per la Ricerca sul Cancro (AIRC) IG 10376, Carraresi Foundation and by Legge 7 Regione Autonoma Sardegna (R.A.S). There are no conflicts of interest. © The Author 2013
Use of a micro-bioreactor to promote 3-dimensional cell rearrangement and induce, maintain, and stabilize high plasticity in epigenetically erased fibroblasts
No full textDevelopment 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
No shortcuts to pig embryonic stem cells
No full textThe establishment of embryonic stem cell (ESC) lines in domestic species could have great impact in the agricultural as well as in the biomedical field. In particular, derivation of pig ESC would find important applications aimed at improving health and production traits of this species through genetic engineering. Similarly, the immunological, morphological, physiological, and functional similarities to the human make the pig a very effective and suitable animal model for biomedical studies and pre-clinical trials. While proven blastocyst-derived mouse and human ESC lines have been established, no validated porcine ESC (pESC) lines are available. In the present manuscript we briefly discuss some of the factors that make the establishment of ESC lines in the pig, and in animal species other than mouse and human, a very slow process. The paucity of information related to morphology, pluripotency markers, differentiation capability hampers a thorough evaluation of the validity of putative lines. These difficulties are further increased by the lack of reliable antibodies, reagents, and in vitro culture systems that could ensure reliable results in the pig and allow for the screening and long-term maintenance of pESC. Data from the literature suggest that similar regulatory pathways are likely to exist among different species. Coupling of these pathways with their distinct expression patterns, the relative concentrations of pluripotency-related molecules, and timing of embryo development, along with supportive micro-environmental conditions, would appear to vary in a species-specific manner. We feel that the understanding of these subtle but meaningful diversities may provide beneficial information about the isolation of genuine porcine embryonic stem cells
Parthenogenesis in mammals: pros and cons in pluripotent cell derivation
No full textEmbryonic stem cells (ESCs) represent a useful tool for cell therapy studies, however the use of embryos for their derivation give rise to ethical, religious and legal problems when applied to the human. During the last years parthenogenesis has been proposed as an alternative source to obtain ESCs. Based on the fact that parthenotes avoid many concerns surrounding the "ad hoc" in vitro production and following destruction of viable human embryos. Unfortunately many aspects related to parthenogenetic cell biology are not fully understood and still need to be elucidated. In this review we describe advantages and limits of these cells. We discuss their typical ESC morphology and high telomerase activity, which disappears after differentiation. We examine the pluripotency signature that they share with bi-parental ESCs. We review their high differentiation plasticity that allow for the derivation of several mature cell type populations when we expose these cells to adequate conditions. On the other hand, in-depth analysis demonstrated chromosome mal-segregation and altered mechanisms controlling centriole arrangement and mitotic spindle formation in these cells. We hypothesize their monoparental origin as one of the possible cause of these anomalies and suggest a great caution if a therapeutic use is considered
Mountain high and valley deep: epigenetic controls of pluripotency and cell fate
No full textAll 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
Epigenetic Conversion as a Safe and Simple Method to Obtain Insulin-secreting Cells from Adult Skin Fibroblasts
No full textRegenerative medicine requires new, fully functional cells that are delivered to patients in order to repair degenerated or damaged tissues. When such cells are not readily available, they can be obtained using different approaches that include, among the many, reprogramming and trans-differentiation, with advantages and limitations that are specific of the different techniques. Here a new strategy for the conversion of an adult mature fibroblast into an insulin-secreting cell, arbitrarily designated as epigenetic converted cells (EpiCC), is described. The method has been developed, based on the increasing understanding of the mechanisms controlling epigenetic regulation of cell fate and differentiation. In particular, the first step uses an epigenetic modifier, namely 5-aza-cytidine, to drive adult cells into a "highly permissive" state. It then takes advantage of this brief and reversible window of epigenetic plasticity, to re-address cells toward a different lineage. The approach is designated "epigenetic cell conversion". It is a simple and robust way to obtain an efficient, controlled and stable cellular inter-lineage switch. Since the protocol does not involve the use of any gene transfection, it is free of viral vectors and does not involve a stable pluripotent state, it is highly promising for translational medicine applications
- …
