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Ramos-Ibeas P.,Laboratory of Reproductive Technologies | Barandalla M.,Laboratory of Reproductive Technologies | Colleoni S.,Laboratory of Reproductive Technologies | Lazzari G.,Laboratory of Reproductive Technologies
Molecular and Cellular Biochemistry | Year: 2017

Oxidative stress has been related to multiple diseases, especially during early embryonic development, when environmental alterations can lead to long-term deleterious effects. In vitro studies of oxidative stress have been mainly focused on somatic cells, but embryonic stem cells (ESCs) represent a promising model of early embryonic development as they are the in vitro equivalent to pluripotent cells in the embryo. Human fibroblasts and ESCs were exposed to different pro-oxidant agents (hydrogen peroxide, tert-butyl hydroperoxide (TBHP), and rotenone) and antioxidants (sodium pyruvate, N-acetylcysteine, Trolox, and sodium selenite) during a 72 h oxidative stress treatment. Then, cell viability, oxidative stress, mitochondrial activity, and gene expression were analyzed, focusing on the antioxidant effect of pyruvate. Pyruvate protected both somatic and pluripotent cells against different pro-oxidant agents, showing strong ROS scavenging capacity, protecting mitochondrial membrane potential, and regulating gene expression and cell metabolism through different mechanisms in fibroblasts and ESCs. In fibroblasts, pyruvate avoided NFKβ nuclear translocation and the upregulation of genes related to the oxidative stress response, while in ESCs pyruvate stimulated the expression of genes involved in anaerobic glycolysis. Fibroblasts and ESCs reacted in different ways to oxidative stress and antioxidant treatment, and pyruvate was the most complete antioxidant, protecting both cell types at different levels. © 2017, Springer Science+Business Media New York.


Busby S.-A.,Glasgow Caledonian University | Crossan C.,Glasgow Caledonian University | Godwin J.,Glasgow Caledonian University | Petersen B.,Friedrich Loeffler Institute | And 5 more authors.
Xenotransplantation | Year: 2013

The hepatitis E virus (HEV) is considered a zoonotic pathogen. In xenotransplantation, given the high prevalence of HEV infection in pigs, the risk of zoonotic transmission from a porcine source is considered high. Currently no clear data are available on how to diagnose and eliminate HEV in herds used for medical purposes and the importance of viral infection at the stage of harvest. In this study, several groups of animals currently used for medical purposes were found RNA positive in both serum and faeces for HEV genotype 3. In addition, viraemia was found in animals up to 3.6 yr of age, which is much longer than originally expected. Herd transmission rates appeared to be significantly lower in animals kept under minimal barrier conditions, compared with those observed for commercial animals, and as expected, segregation of animals at an early age prevented spread of infection. This study makes suggestions to ensure appropriate detection and eradication of HEV from a donor herd to be used for xenotransplantation purposes. © 2013 John Wiley & Sons A/S.


Rossi B.,University of Bologna | Merlo B.,University of Bologna | Colleoni S.,Laboratory of Reproductive Technologies | Iacono E.,University of Bologna | And 5 more authors.
Stem Cell Reviews and Reports | Year: 2014

Amniotic fluid (AF) is a source of multipotent mesenchymal stem cells (MSCs), very promising cells for tissue engineering in clinical application. The aim of this work was to isolate and characterize cells isolated from bovine AF as alternative sources of primitive multipotent stem cells in a species that could be a large-animal model for biomedical and biotechnology researches. Samples were recovered, at slaughterhouse, from 39 pregnant cows at different trimesters of pregnancy and cells were cultured in vitro. At passages (P) 3 and 7 differentiation towards chondrogenic, osteogenic and adipogenic lineages was induced. Flow cytometry analysis for CD90, CD105, CD73, CD44, CD34, CD45 and CD14 was performed, immunocytochemistry (ICC) for Oct4, SSEA4, α-SMA, Vimentin, N- and E- Cadherin and CK and qPCR analysis for OCT4, NANOG and SOX2 were carried out. The cell yield was significantly higher in the first trimester compared to the second and the third one (P < 0.05). Cells were isolated from 25/39 samples and cell population appeared heterogeneous. Two main cell types were identified in samples from all trimesters: round- (RS) and spindle-shaped (SS) cells. 17/25 samples showed both populations (mixed, MX). Both cell types showed MSC-markers and differentiation capability with some variability related to the passages. The SS-population also expressed low levels of stemness markers such as NANOG and SSEA4 but not OCT4. Bovine AF shows a heterogeneous cell population containing also MSCs, multipotent cells that represent an intermediate stage between embryonic stem cells and adult ones. © 2014, Springer Science+Business Media New York.


Lauri A.,Parco Tecnologico Padano | Lazzari G.,Laboratory of Reproductive Technologies | Galli C.,Laboratory of Reproductive Technologies | Galli C.,University of Bologna | And 5 more authors.
Genomics | Year: 2013

The possibility to genotype embryos prior to implantation would have advantages for increasing the speed of selection of cattle. Reliable genotyping requires more DNA than can be obtained from biopsies of embryos, if they are to remain viable. Multiple displacement amplification (MDA) is a whole genome amplification technique used to increase the amount of DNA from biopsies for analysis. Reduced genome coverage resulting in Allele Drop Out (ADO) at heterozygous loci or missing genotypes are drawbacks of MDA.The present article describes the correlation between the input DNA quantity or embryo biopsy size and MDA success. Missing genotypes and ADO drastically increased when fewer than 30-40 cells or the genomic equivalents were used. However, embryo viability was found to be reduced if biopsied with more than 10 cells. Therefore, in vitro cell culture was investigated as a means to increase the number of cells available and the genotyping reliability. © 2012 Elsevier Inc.


PubMed | University of Manitoba, University Claude Bernard Lyon 1, French Institute of Health and Medical Research, Laboratory of Reproductive Technologies and Xenothera
Type: Journal Article | Journal: PloS one | Year: 2016

Polyclonal xenogenic IgGs, although having been used in the prevention and cure of severe infectious diseases, are highly immunogenic, which may restrict their usage in new applications such as Ebola hemorrhagic fever. IgG glycans display powerful xenogeneic antigens in humans, for example 1-3 Galactose and the glycolyl form of neuraminic acid Neu5Gc, and IgGs deprived of these key sugar epitopes may represent an advantage for passive immunotherapy. In this paper, we explored whether low immunogenicity IgGs had a protective effect on a guinea pig model of Ebola virus (EBOV) infection. For this purpose, a double knock-out pig lacking 1-3 Galactose and Neu5Gc was immunized against virus-like particles displaying surface EBOV glycoprotein GP. Following purification from serum, hyper-immune polyclonal IgGs were obtained, exhibiting an anti-EBOV GP titer of 1:100,000 and a virus neutralizing titer of 1:100. Guinea pigs were injected intramuscularly with purified IgGs on day 0 and day 3 post-EBOV infection. Compared to control animals treated with IgGs from non-immunized double KO pigs, the anti-EBOV IgGs-treated animals exhibited a significantly prolonged survival and a decreased virus load in blood on day 3. The data obtained indicated that IgGs lacking 1-3 Galactose and Neu5Gc, two highly immunogenic epitopes in humans, have a protective effect upon EBOV infection.


Dieci C.,Laboratory of Reproductive Technologies | Lodde V.,Laboratory of Reproductive Technologies | Lodde V.,University of Milan | Franciosi F.,University of Milan | And 8 more authors.
Biology of Reproduction | Year: 2013

In the pig, the efficiency of in vitro embryo production and somatic cell nuclear transfer (SCNT) procedures remains limited. It has been suggested that prematuration treatments (pre-IVM) based on the prolongation of a patent, bidirectional crosstalk between the oocyte and the cumulus cells through gap junction mediate communication (GJC), with the maintenance of a proper level of cAMP, could improve the developmental capability of oocytes. The aim of this study was to assess: 1) dose-dependent effects of cilostamide on nuclear maturation kinetics, 2) the relationship between treatments on GJC functionality and large-scale chromatin configuration changes, and 3) the impact of treatments on developmental competence acquisition after parthenogenetic activation (PA) and SCNT. Accordingly, cumulus-oocyte complexes were collected from 3- to 6-mm antral follicles and cultured for 24 h in defined culture medium with or without 1 lM cilostamide. GJC functionality was assessed by Lucifer yellow microinjection, while chromatin configuration was evaluated by fluorescence microscopy after nuclear staining. Cilostamide administration sustained functional coupling for up to 24 h of culture and delayed meiotic resumption, as only 25.6% of cilostamide-treated oocytes reached the pro-metaphase I stage compared to the control (69.7%; P < 0.05). Moreover, progressive chromatin condensation was delayed before meiotic resumption based upon G2/M biomarker phosphoprotein epitope acquisition using immunolocalization. Importantly, cilostamide treatment under these conditions improved oocyte developmental competence, as reflected in higher blastocyst quality after both parthenogenetic activation and SCNT. © 2013 by the Society for the Study of Reproduction, Inc.


Hall V.,Copenhagen University | Hinrichs K.,Texas A&M University | Lazzari G.,Laboratory of Reproductive Technologies | Betts D.H.,University of Western Ontario | Hyttel P.,Copenhagen University
Veterinary Journal | Year: 2013

Over many decades assisted reproductive technologies, including artificial insemination, embryo transfer, in vitro production (IVP) of embryos, cloning by somatic cell nuclear transfer (SCNT), and stem cell culture, have been developed with the aim of refining breeding strategies for improved production and health in animal husbandry. More recently, biomedical applications of these technologies, in particular, SCNT and stem cell culture, have been pursued in domestic mammals in order to create models for human disease and therapy. The following review focuses on presenting important aspects of pre-implantation development in cattle, pigs, horses, and dogs. Biological aspects and impact of assisted reproductive technologies including IVP, SCNT, and culture of pluripotent stem cells are also addressed. © 2013 Elsevier Ltd.


Colleoni S.,Laboratory of Reproductive Technologies | Galli C.,Laboratory of Reproductive Technologies | Galli C.,University of Bologna | Gaspar J.A.,Institute of Neurophysiology | And 5 more authors.
Current Medicinal Chemistry | Year: 2012

The development of in vitro testing strategies for chemical and drug screening is a priority need in order to protect human health, to increase safety, to reduce the number of animals required for conventional testing methods and finally to meet the deadlines of current legislations. The aim of this work was to design an alternative testing method based on human embryonic stem cells for the detection of prenatal neural toxicity. For this purpose we have created a model based on the generation of neural rosettes, reproducing in vitro the gastrulation events recapitulating the formation of the neural tube in vivo. To validate the model we have exposed this complex cell system to increasing concentrations of valproic acid, a known teratogenic agent, to analyse the morphological and molecular changes induced by the toxicant. Specific assays were applied to discriminate between cytotoxicity and specific neural toxicity. Transcriptomic analysis was performed with a microarray Affimetrix platform and validated by quantitative real time RT-PCR for the expression of genes involved in early neural development, neural tube formation and neural cells migration, key biological processes in which the effect of valproic acid is most relevant. The results demonstrated that neural rosette cells respond to valproic acid exposure with molecular and morphological changes similar to those observed in vivo, indicating that this method represents a promising alternative test for the detection of human prenatal neural toxicity. © 2012 Bentham Science Publishers.


Sartori C.,Laboratory of Reproductive Technologies | Didomenico A.I.,Roslin Institute | Thomson A.J.,Roslin Institute | Milne E.,Roslin Institute | And 3 more authors.
Cellular Reprogramming | Year: 2012

Pluripotential stem cells from livestock offer an exciting prospect for the biotechnology industry. Applying strategies established for the derivation of murine induced pluripotential stem cells (iPSCs), we have isolated ovine iPSCs that can give rise to cells characteristic of all three germ cell layers both in vitro from embryoid bodies and in teratomas in vivo. Furthermore, although at a low level, these ovine iPS cells can contribute to live-born chimeric lambs. Colonies derived from ovine embryonic fibroblasts transfected with murine cMyc, Klf4, Oct4, and Sox2 displayed smooth domes with sharp edges when grown in human embryonic stem cell (ESC) medium but not in mouse ESC medium. These ovine iPSCs were alkaline phosphatase positive, expressed Nanog, and had a normal karyotype. These cells represent an important step in the understanding of mechanistic nature of pluripotency in ungulates. © 2012, Mary Ann Liebert, Inc.


Galli C.,Laboratory of Reproductive Technologies | Galli C.,University of Bologna | Duchi R.,Laboratory of Reproductive Technologies | Colleoni S.,Laboratory of Reproductive Technologies | And 2 more authors.
Theriogenology | Year: 2014

Assisted reproductive techniques developed for cattle in the last 25 years, like ovum pick up (OPU), intracytoplasmic sperm injection (ICSI), and somatic cell nuclear transfer, have been transferred and adapted to buffalo and horses. The successful clinical applications of these techniques require both the clinical skills specific to each animal species and an experienced laboratory team to support the invitro phase of the work. In cattle, OPU can be considered a consolidated technology that is rapidly outpacing conventional superovulation for embryo transfer. In buffalo, OPU represents the only possibility for embryo production to advance the implementation of embryo-based biotechnologies in that industry, although it is still mainly in the developmental phase. In the horse, OPU is now an established procedure for breeding from infertile and sporting mares throughout the year. It requires ICSI that in the horse, contrary to what happens in cattle and buffalo, is very efficient and the only option because conventional IVF does not work. Somatic cell nuclear transfer is destined to fill a very small niche for generating animals of extremely high commercial value. The efficiency is low, but because normal animals can be generated it is likely that advancing our knowledge in that field might improve the technology and reduce its cost. © 2014 Elsevier Inc.

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