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Schubert T.,Catholic University of Louvain | Poilvache H.,Catholic University of Louvain | Galli C.,Laboratorio Of Tecnologie Della Riproduzione | Galli C.,University of Bologna | And 2 more authors.
Biomaterials | Year: 2013

Pig adipose mesenchymal stem cells (AMSCs) could be proposed for the improvement of bone substitute. However, these xenogenic cells retain a galactosyl (Gal) epitope that elicits xenorejection. Our work aims to use Gal-Knock-Out (Gal-KO) pig AMSCs to associate cellular immunomodulation, humoral down-elicitation of Gal-KO cells and osteogenic capacity of AMSCs. Human and pig AMSCs were compared for proliferation/differentiation kinetics and bone neoformation in vivo. Humoral reaction against pig Gal+ vs. Gal-KO AMSCs and immunomodulation properties of Gal+ vs. Gal-KO AMSCs were assessed in vitro. Humoral/cellular reactions against Gal+ vs. Gal-KO osteogenic differentiated pig AMSC xenografts were assessed in an immunocompetent rodent model. Expansion/differentiation/bone neoformation was significantly improved with differentiated pig AMSCs compared with human cells. Based on immunohistochemistry and cell-based ELISA, Gal+ AMSCs had higher sensitivity to preformed/induced anti-pig antibodies than Gal-KO AMSCs. In vitro cellular immunomodulation was similar between Gal+ and Gal-KO AMSCs. In vivo, a significant reduction of anti-pig IgG was found at 1 month in rats implanted with Gal-KO AMSCs compared with those implanted with Gal+ AMSCs. Lymphocyte/macrophage infiltration of osteogenic differentiated pig AMSC xenografts was significantly lower at post-operative day (POD) 7 in recipients of Gal-KO vs. Gal+ pig cells. No significant difference was found at POD 28. The combination of the cellular immunomodulation with the Gal-KO phenotype of AMSCs can significantly improve the cellular engraftment of pig osteogenic cells by delaying xenorejection. © 2013 Elsevier Ltd.

Byrne M.J.,IDI Group | Zatta A.,IDI Group | Galli C.,Laboratorio Of Tecnologie Della Riproduzione | Power J.M.,IDI Group | Kaye D.M.,IDI Group
Journal of Interventional Cardiology | Year: 2010

Introduction: Despite pharmacological advances for heart failure, morbidity and mortality remain unacceptably high. As a result, alternative approaches such as cell therapy have been suggested to hold potential promise. However, a major obstacle is the optimization of cell delivery to the heart. Therefore, we investigated the efficacy of a percutaneous recirculation system for the delivery of cells to the heart. Methods: Ovine fibroblasts were delivered to the ovine heart (3 × 107 cells) using the V-Focus system, a "closed" recirculatory system that draws blood from the coronary sinus and returns it to the coronary artery via an oxygenator, or intracoronary (IC) infusion, followed by a 2-hour recovery period. Animals were euthanized and cardiac tissue collected to determine presence of cells. Results: There was a significant difference (P < 0.05) in the number of cells delivered to the heart by the V-Focus compared to direct coronary infusion for left ventricular freewall (V-Focus 1.39 ± 0.63/mm2, IC 0.11 ± 0.06/mm2), septum (V-Focus 3.18 ± 0.88/mm2, IC 0.38 ± 0.19/mm2), and right ventricle (V-Focus 0.46 ± 0.23/mm2, IC 0.05 ± 0.04/mm2). Conclusions: These results suggest that potential therapeutic cells are optimally delivered to the large animal heart using the V-Focus cardiac delivery system in an ovine heart. © 2009, Wiley Periodicals, Inc.

Hendriks W.K.,University Utrecht | Hendriks W.K.,Repro | Colleoni S.,Laboratorio Of Tecnologie Della Riproduzione | Galli C.,Laboratorio Of Tecnologie Della Riproduzione | And 7 more authors.
Reproduction, Fertility and Development | Year: 2015

Advanced maternal age and in vitro embryo production (IVP) predispose to pregnancy loss in horses. We investigated whether mare age and IVP were associated with alterations in mitochondrial (mt) DNA copy number or function that could compromise oocyte and embryo development. Effects of mare age (<12 vs ≥12 years) on mtDNA copy number, ATP content and expression of genes involved in mitochondrial replication (mitochondrial transcription factor (TFAM), mtDNA polymerase γ subunit B (mtPOLB) and mitochondrial single-stranded DNA-binding protein (SSB)), energy production (ATP synthase-coupling factor 6, mitochondrial-like (ATP-synth-F6)) and oxygen free radical scavenging (glutathione peroxidase 3 (GPX3)) were investigated in oocytes before and after in vitro maturation (IVM), and in early embryos. Expression of TFAM, mtPOLB and ATP-synth-F6 declined after IVM (P<0.05). However, maternal age did not affect oocyte ATP content or expression of genes involved in mitochondrial replication or function. Day 7 embryos from mares ≥12 years had fewer mtDNA copies (P≤0.01) and lower mtDNA:total DNA ratios (P<0.01) than embryos from younger mares, indicating an effect not simply due to lower cell number. Day 8 IVP embryos had similar mtDNA copy numbers to Day 7 in vivo embryos, but higher mtPOLB (P≤0.013) and a tendency to reduced GPX3 expression (P≤0.09). The lower mtDNA number in embryos from older mares may compromise development, but could be an effect rather than cause of developmental retardation. The general down-regulation of genes involved in mitochondrial replication and function after IVM may compromise resulting embryos. © CSIRO 2015.

Arabi S.,Istituto Sperimentale Italiano Lazzaro Spallanzani | Balduzzi D.,Istituto Sperimentale Italiano Lazzaro Spallanzani | Bechi N.,University of Siena | Cordelli E.,ENEA | And 10 more authors.
Annali dell'Istituto Superiore di Sanita | Year: 2011

Reproductive toxicity, with its many targets and mechanisms, is a complex area of toxicology; thus, the screening and identification of reproductive toxicants is a main scientific challenge for the safety assessment of chemicals, including the European Regulation on Chemicals (REACH). Regulatory agencies recommend the implementation of the 3Rs principle (refinement, reduction, replacement) as well as of intelligent testing strategies, through the development of in vitro methods and the use of mechanistic information in the hazard identification and characterization steps of the risk assessment process. The EU Integrated Project ReProTect (6 th Framework Programme) implemented an array of in vitro tests to study different building blocks of the mammalian reproductive cycle: methodological developments and results on male and female germ cells, prostate and placenta are presented.

Galli C.,Laboratorio Of Tecnologie Della Riproduzione | Galli C.,University of Bologna | Perota A.,Laboratorio Of Tecnologie Della Riproduzione | Brunetti D.,Laboratorio Of Tecnologie Della Riproduzione | And 3 more authors.
Xenotransplantation | Year: 2010

Background: Techniques for genetic engineering of swine are providing genetically modified animals of importance for the field of xenotransplantation, animal models for human diseases and for a variety of research applications. Many of these modifications have been directed toward avoiding naturally existing cellular and antibody responses to species-specific antigens. Methods: A number of techniques are today available to engineering the genome of mammals, these range from the well established less efficient method of DNA microinjection into the zygote, the use of viral vectors, to the more recent use of somatic cell nuclear transfer. The use of enzymatic engineering that are being developed now will refine the precision of the genetic modification combined with the use of new vectors like transposons. Results: The use of somatic cell nuclear transfer is currently the most efficient way to generate genetically modified pigs. The development of enzymatic engineering with zinc-finger nucleases, recombinases and transposons will revolutionize the field. Nevertheless, genetic engineering in large domesticated animals will remain a challenging task. Conclusions: Recent improvements in several fields of cell and molecular biology offer new promises and opportunities toward an easier, cost-effective and efficient generation of transgenic pigs. © 2010 John Wiley & Sons A/S.

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