1,721,045 research outputs found
CENTROSOME BIOGENESIS AND ADAPTIVE RESPONSE IN MAMMAL PARTHENOGENETIC CELLS
Full text linkHuman 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
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
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
Mountain high and valley deep: epigenetic controls of pluripotency and cell fate
Full text linkAll 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
In vitro viability of sheep whole ovaries and cortical fragments after cryopreservation with different techniques
No full textCryopreservation of ovarian tissue is a promising technique for preserving fertility in young female cancer patients. At present two options are available: cryopreservation of ovarian cortical fragments or of the whole ovary. We compared ovarian tissue viability, cryopreserved as fragments or whole organs using a conventional (CF) or directional freezing apparatus (DF).
Cortical fragments (10x5x1 mm) were immersed into Leibovitz L-15 medium, 10% FCS and 1,5 M DMSO, while whole ovaries were perfused with the same solution. CF was performed at 0.5°C/min in a Kryo 560M (Planer, UK). DF was performed at 0.01 mm/sec, resulting in cooling rates of 0.3°C/min with a Multi-Thermal-Gradient (IMT, Israel). In both cases freezing was arrested at -40°C, before plunging the samples into liquid nitrogen. After thawing, whole ovaries and cortical fragments were cut in 2x2x1 mm pieces and cultured for 7 days in α-MEM medium supplemented with ITS, glutamine, pyruvate, hypoxantine, BSA, FSH and bFGF. After culture, the percentage of primordial follicles developed to the primary stage in DF whole ovaries, was comparable to controls. A lower rate (P<0.05) of growing follicles was observed in all other groups, and CF whole ovaries were unable to support any development. DNA double-strand breaks formation and repair was analyzed by immunofluorescent expression of γ-H2AX and of RAD51. At the beginning of the culture, DNA damage in frozen samples was higher than in fresh controls. However after 7 days, DNA damage significantly decrease in DF groups and in CF cortex samples concomitantly with an increase of DNA repair. Conversely, in CF whole ovaries DNA repair signal was absent leaving the rate of DNA damage substantially unchanged. We conclude that DF allows a better preservation than CF of whole ovaries and cortical fragments and eliminates any disadvantage related to the bigger volume of whole organs
Pluripotency network in porcine embryos and derived cell lines
No full textHuge amounts of work have been dedicated to the establishment of embryonic stem cell lines from farm animal species since the successful isolation of embryonic stem cells from the mouse and from the human. However, no conclusive results have been obtained so far, and validated lines have yet to be established in domestic animals. Many limiting factors have been suggested and need to be studied further to isolate truly pluripotent cell lines from livestock. In this review, we will discuss the difficulties in deriving and maintaining embryonic stem cell lines from farm animal embryos and how can this lack of success be explained. We will summarize results obtained in our laboratory regarding derivation of pluripotent cells in the pigs. Problems related to the identification of standard methods for derivation, maintenance and characterization of cell lines will also be examined. We will focus our attention on the need for appropriate stemness-related marker molecules that can be used to reliably investigate pluripotency in domestic species. Finally, we will review data presently available on functional key pluripotency-maintaining pathways in farm animals
Multi-thermal gradient freezing allows the cryopreservation of sheep whole ovaries with the same efficiency of ovarian fragments
No full textOvarian tissue cryobanking is proposed as an effective option for preserving female fertility in cancer patients. At present 2 options are available: cryopreservation of ovarian cortical fragments or of the whole ovary. The use of whole ovary reduces ischemic insult. However, the larger the sample volume, the more difficult it is to introduce the cryoprotective agents and to ensure an adequate cooling rate that minimizes tissue damage. For this reason, we used the multi-thermal gradient method, based on running the sample through a temperature gradient. This allows a homogeneous cooling rate through the whole sample independently from its volume. The aim of the study was to determine whether multi-thermal gradient freezing allows a substantial reduction of the damages induced by cryopreservation of large samples by comparing the viability of cortical fragments versus whole ovaries after thawing and grafting in nude mice. Sheep ovaries were collected at the local abattoir and randomly divided into 3 groups: A) ovaries frozen as cortical fragments, B) ovaries frozen as whole organs, and C) fresh ovaries immediately processed for further analysis (control). Ovarian fragments (10×5×1mm) were sliced from the cortical region and immersed into cryoprotectant solution (Leibovitz L-15 medium, 10% FCS, and 1.5M dimethyl sulfoxide), while whole ovaries were perfused with the same solution. Samples were placed into glass freezing tubes 16mm in diameter filled with cryoprotectant solution. Samples were frozen with the multi-thermal gradient freezing apparatus (Core Dynamics, Ness Ziona, Israel) progressing along the thermal gradient at a rate of 0.01mms(-1), resulting in a cooling rate of 0.3°Cmin(-1). Two weeks later, samples were thawed by plunging the tubes into a 37°C water bath with gentle shaking. Whole ovaries were perfused with 10mL of HEPES-Talp medium, 0.5M sucrose, and 10IUmL(-1) of heparin and their cortical region was cut into fragments. These fragments and those derived from group A were rehydrated in L-15 medium with decreasing sucrose concentrations. Fragments (2×2×1mm) were xenografted in the dorsal region of 6 nude mice for each group. Mice were killed after 8 weeks and grafts were collected for analysis. Cryopreserved samples were compared with each other and fresh controls (group C). Morphologically normal follicles at primordial, primary, and secondary stages were visible in all samples. Cell proliferation was assessed measuring Ki-67 mRNA and counting immunohistochemically positive cells. The FSH receptor and GDF9 gene expression were used to evaluate tissue viability. No significant differences for any of these parameters were measured amongst the groups. We conclude that directional freezing is an effective method for ovarian tissue cryopreservation independently from the sample volume, thus overriding the limitations usually associated with whole-organ banking
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