Cesare Galli

University of Bologna, Bolonia, Emilia-Romagna, Italy

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Publications (147)426.11 Total impact

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    ABSTRACT: The zona-free method of SCNT designed for bovine and pig cloning (Booth et al. 2001; Vajta et al. 2001; Oback et al. 2003) was successfully used for horse (Galli et al. 2003). Although simple and efficient in farm animals, its application in the mouse met several problems (Ribas et al. 2005, 2006). The aim of our work was to produce cloned mice using HM1 embryonic stem (ES)cells adapting a zona-free method. Seven- to 24-week-old superovulated B6D2F1 female mice were used as oocytes donors. Cumulus cells were removed by 0.3% hyaluronidase and the zona pellucida by 0.5% pronase in KSOM-HEPES (KSOM-H) 1h later (Ribas et al. 2006) or immediately after hyaluronidase treatment at 37°C. The HM1 ES cells were cultured in KnockOut DMEM supplemented with leukemia inhibitory factor and 15% fetal bovine serum with or without 2i (Ying et al. 2008) and were synchronized at M phase by 3ngmL(-1) nocodazole for 3h before fusion. Only spherical cells were selected for NT. Metaphase II chromosome spindle complexes were removed by micromanipulation in KSOM-H medium with 5μgmL(-1) cytochalasin B. Lectin-treated enucleated oocytes were attached to the donor cells in KSOM-H with nocodazole and fused by 2 pulses of 1.3kVcm(-1) DC for 30μs in 0.3M mannitol medium. Following 10- to 15-min incubation in KSOM-H, the fusion was assessed and repeated if the constructs were nonfused. Cloned embryos were activated in 1mM SrCl2 in Ca(2+)-free KSOM medium for 2 to 2.5 or 5 to 6h and cultured in 20-μL KSOM droplets using the well-of-the-well (WOW) method (Vajta et al. 2000) under mineral oil at 37°C and 5% CO2. Day 4 compacted morulae and blastocysts were surgically transferred into the uterus of Day-2.5 pseudopregnant recipients that were sacrificed on Day 19.5 to examine fetal development. The donor mice age was important for oocyte survival: ~16% of oocytes of 7- to 10-week-old mice lysed before or during fusion in 33% of experiments (n experiments=15), whereas oocytes of older mice were not sensitive to enzymatic treatment and electric impulses even after 3 fusion rounds (n=19). The time of pronase treatment did not affect oocyte survival, whereas extending the time between hyaluronidase treatment and enucleation revealed self-activation in ~25% of oocytes. The fusion efficiency of ES cells was significantly lower compared with serum-starved fibroblasts (61%, n=623 v. 100%, n=80). The duration of SrCl2 treatment did not affect embryo development (cleavage: 82% v. 84%; Day 4 blastocysts: 49% v. 52%). ES cell culture with 2i increased Day 4 blastocyst development (60.7% v. 50.4%; P=0.07), and their ability to implant (52.6% v. 38.2%; P=0.06). Moreover, only NT embryos derived from 2i-ES cells developed to term (8.2%, n=5; P=0.08), and produced live fetuses (4.9%, n=3). In light of these results, the fusion of ES cells remains the critical step in the mouse zona-free protocol.
    Reproduction Fertility and Development 12/2014; 27(1):113. · 2.58 Impact Factor
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    ABSTRACT: Intracytoplasmic sperm injection (ICSI) is used for assisted fertilization of equine oocytes. However, not all oocytes cleave after ICSI. Maternal aging deleteriously affects fertility in mares and women, with reduced oocyte quality and success of assisted reproductive technologies. In the oocyte, senescence and cell-programmed death begins after maturation; the extent that maternal age affects these events is unknown. We hypothesised that formation of α/β tubulin asters and f-actin bubbles are associated with aging of the oocyte in vitro and/or aging of the oocyte in vivo, in aged donors. In Exp 1, oocytes were collected from ovaries obtained from an abattoir and matured for 28h and selected for polar body extrusion (0h). At 0, 24, and 48h, oocytes (n=38 total) were fixed in MTSB-XF and transferred into wash solution with 1% BSA and 0.1% Triton X-100 in PBS for immunostaining. For experiment 2, oocytes were collected from preovulatory follicles of mares (9-25 yr) in a clinical ICSI program and injected with sperm from various stallions after extrusion of a polar body. Between 24 to 51h after ICSI, uncleaved oocytes (n=52, single cell without evidence of fragmentation or indentation of the oolemma) were fixed. All oocytes were incubated with α/β tubulin and human-anti-centromere antibody-CREST/ACA (1:100 each). Following primary incubation, oocytes were washed and incubated with Alexa 488, Alexa 647, Alexa 561-phalloidin, and Hoechst 33258. Images and Z-stacks were acquired on an Olympus IX81 spinning disk confocal microscope. Morphometric and intensity analyses of images were performed using SlideBook software (Denver, CO). Student's t-test, Fisher's exact test, and chi-square analyses were used for statistical comparisons. After aging in vitro (experiment 1), the number of oocytes with tubulin multiasters increased (P<0.001; 9% at 0h, 14% at 24h, 85% at 48h); however, actin bubbling was observed in only 5/38 (13%) oocytes, with no effect of incubation time. In experiment 2, tubulin multiasters were present in 62% of oocytes that failed to cleave. More multiasters were observed per oocyte from mares ≤13 yr than ≥20 yr (P=0.03) and fixed at 24 to 28h than 44 to 51h (P=0.04). Actin bubbles were observed in 71% of oocytes that failed to cleave after ICSI, with more actin bubbles in oocytes from mares ≥20 yr than ≤13 yr (P=0.01) and fixed 44 to 51h versus 24 to 28h after ICSI (P=0.05). The sum intensity and area of the actin bubbles were higher in oocytes fixed at 44 to 51h than 24 to 28h (P=0.01 and P=0.04). The area occupied by the actin bubbles was larger (P=0.05) in oocytes from mares ≥20 yr than ≤13 yr. This study demonstrates actin bubbles and tubulin asters are involved in oocyte aging and cytoskeleton remodelling with or without fertilization. Although actin structures were associated with donor age and hours after ICSI, they were not present in unfertilized oocytes aged in vitro. Multiaster formation was associated with cell senescence in oocytes aged in vitro. Although not previously reported for the equine oocyte, multiaster formation appeared to be an initial fertilization event within the oocyte associated with attempted zygote development.
    Reproduction Fertility and Development 12/2014; 27(1):249-50. · 2.58 Impact Factor
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    ABSTRACT: A number of in vitro toxicity assays based on human embryonic stem cells (hESCs) are under development in order to provide alternative methods for the screening of chemicals and drugs and to reduce the number of animals needed for developmental toxicity assessment. The major challenge is to demonstrate the reliability of these in vitro methods by correlating the in vitro produced results to the available in vivo data. In this context transcriptomic approaches associated to toxicogenomic database analysis give the possibility to screen, annotate and cluster high numbers of genes and to identify the molecular changes that univocally mark the toxicity induced processes or are indicative of the early initiating events that lead to cellular toxicity. In this retrospective study we compare microarray transcriptomic data derived from two different hESCs lines (HUES1 and H9) exposed to valproic acid (VA) while applying the same differentiation protocol. We present the results of this comparative analysis in light of the known teratogenic effects of VA. The results show molecular changes in the processes of neural development, neural crest migration, apoptosis and regulation of transcription, indicating a good correspondence with the available in vivo data. We also describe common toxicological signatures and provide an interpretation of the observed qualitative differences referring to known biological features of the two hESCs lines.
    Toxicology Letters 09/2014; · 3.15 Impact Factor
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    ABSTRACT: Background Human corneal allografting is an established procedure to cure corneal blindness. However, a shortage of human donor corneas as well as compounding economic, cultural, and organizational reasons in many countries limit its widespread use. Artificial corneas as well as porcine corneal xenografts have been considered as possible alternatives. To date, all preclinical studies using de-cellularized pig corneas have shown encouraging graft survival results; however, relatively few studies have been conducted in pig to non-human primate (NHP) models, and particularly using genetically engineered donors.Methods In this study, we assessed the potential benefit of using either hCTLA4-Ig transgenic or α1,3-Galactosyl Transferase (GT) Knock-Out (KO) plus transgenic hCD39/hCD55/hCD59/fucosyl-transferase pig lines in an anterior lamellar keratoplasty pig to NHP model.ResultsCorneas from transgenic animals expressing hCTLA4-Ig under the transcriptional control of a neuron-specific enolase promoter showed transgene expression in corneal keratocytes of the stroma and expression was maintained after transplantation. Although a first acute rejection episode occurred in all animals during the second week post-keratoplasty, the median final rejection time was 70 days in the hCTLA4-Ig group vs. 21 days in the wild-type (WT) control group. In contrast, no benefit for corneal xenograft survival from the GTKO/transgenic pig line was found. At rejection, cell infiltration in hCTLA4Ig transgenic grafts was mainly composed of macrophages with fewer CD3+ CD4+ and CD79+ cells than in other types of grafts. Anti-donor xenoantibodies increased dramatically between days 9 and 14 post-surgery in all animals.Conclusions Local expression of the hCTLA4-Ig transgene dampens rejection of xenogeneic corneal grafts in this pig-to-NHP lamellar keratoplasty model. The hCTLA4-Ig transgene seems to target T-cell responses without impacting humoral responses, the control of which would presumably require additional peripheral immunosuppression.
    Xenotransplantation 07/2014; · 2.57 Impact Factor
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    ABSTRACT: 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.
    Stem cell reviews. 06/2014;
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    ABSTRACT: Objective: To investigate the telomere length in bovine offspring produced by a cloned and control bull, and the telomerase activity in embryos produced with the same technology. Methods: Five daughters of a control and five daughters of a bull cloned using a fibroblast of the control were produced by IVF using sperm of the two bulls. Blood samples of the offspring were collected at 2, 6, and 12 months of age and the relative telomere length (RTL) was assessed by flow cytometry. At same time the body growth, hematological profile, and clinical biochemistry of the same progeny was extensively surveyed, and results have been reported in a previous work. Thereafter, the telomerase activity was assessed using a real time PCR quantitative assay in groups of embryos produced with the same technology. Results: The offspring of the clone exhibited a modest, but significant (P<0.05), shortening of the telomeres (21.36%, 20.56% and 20.56%) compared to that of the CONTROL (23.78%, 23.53% and 22.43%) as mean values determined at 2, 6 and 12 months, respectively. Shortening of telomeres in respect to the age was not significant. No statistical difference was reported between telomerase activity assessed in 144 cloned (3.4-03 ± 2.4-03 amoles/µL) and 80 control (2.1-03 ± 1.8-03 amoles/µL) embryos. Conclusions: The results have revealed a moderate shortening of telomeres in the offspring of the clone with respect to control. However, this study did not evidence differences in the two progenies that suggest welfare problems during the first year of life.
    Asian Pacific Journal of Reproduction. 01/2014; 3(1):1-7.
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    ABSTRACT: The aim of this article was to compare plasma estrone sulfate (E1SO4), clinical biochemistry, and milk yield of dairy cows carrying a female fetus from a bull (BULL) or from its clone (CLONE), evaluating also the relationship between the former variables and the birth weight of the newborn. Sixteen recipient dairy Friesian heifers (10 BULL and 7 CLONE) received a female embryo, obtained by in vitro embryo production and sexing by polymerase chain reaction with the semen of the BULL or the CLONE. Blood samples on all cows were obtained before feed distribution in the morning from jugular vein from 4 weeks before to 4 weeks after calving, to be analyzed for metabolic profile. The samples from late gestation were also analyzed for E1SO4 concentration. To separately assess the effect of calf birth weight (CBW), data were categorized as follows: low (<39 kg; BWT-A), mid (39-46 kg; BWT-B), and high (>46 kg; BWT-C). The plasma concentrations of β-hydroxybutyric acid (BHB, P = 0.019), Na (P = 0.002), Cl (P = 0.026), strong cation-anion balance (P = 0.020), total bilirubin (P = 0.054), and α1-globulin (P = 0.044) were higher in prepartum BULL recipients than those in CLONE, whereas BHB (P = 0.021) and Mg (P = 0.090) were higher in postpartum BULL recipients, while no differences were recorded in the remaining postpartum parameters. The CBW class had significant interaction with week of gestation on antepartum plasma estrone sulfate (P = 0.021), whereas CBW per se affected antepartum plasma BHB (P = 0.021), and nonesterified fatty acids (NEFA; P = 0.011) being higher in BWT-C which also had the lower NEFA concentration during postpartum. Milk yield was unaffected by the sire used, both for quantitative and qualitative aspects. Cows carrying heavier fetus (BWT-C) had a different lactation affected by month compared with the other 2 CBW groups. From these results, there were no differences between BULL and CLONE recipients. Estrone sulfate, BHB, and NEFA may be used to predict CBW and provide different nutritional management during gestation.
    Theriogenology 01/2014; · 2.08 Impact Factor
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    ABSTRACT: 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 in vitro 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.
    Theriogenology 01/2014; 81(1):138–151. · 2.08 Impact Factor
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    ABSTRACT: Recently, site-specific nucleases (zinc-finger nucleases, ZFN; TAL effector nucleases; and CRISPR) emerged as powerful tools for gene modification of different cells types and enhanced green fluorescent protein (EGFP)-specific ZFN were successfully used in the rat (Geurtz et al. 2010) and in the pig (Watanabe et al. 2010; Whyte et al. 2010). Previously (Brunetti et al. 2008 Clon. Stem Cells), we generated an EGFP transgenic porcine line (Verro2GFP) characterised by a single integration of pCAGGS-EGFP cassette, high ubiquitous EGFP expression, Mendelian transgene transmission, and expression in F1. The aim of this work was to modify a transcriptionally active GFP-locus into one suitable for Cre-mediated recombinase-mediated cassette exchange (RMCE), using EGFP-specific ZFN. Homology arms for promoter-less targeting vector were derived from pCAGGS-EGFP vector (promoter fragment=left-homology-arm=LHA; polyA sequence=right-homology-arm=RHA). Cloning floxed (lox2272/lox5171) hygromycin resistance coding sequence between LHA and RHA sequences, we generated the targeting/RMCE vector (pB5'3'Hygro-PL) and its positive control (C+) for PCR set-up (100-1000 plasmid copies). Verro2GFP fibroblasts cultured in DMEM+M199(1:1)+10% FCS, bFGF in 5% CO2, 5% O2, were transfected using Nucleofector (V-024 program). In ZFN-mediated gene targeting, 2μg of each ZFN coding vector (Sigma-CompoZr(®)) and 2μg of pB5'3'Hygro-PL/KpnI vector were used to "nucleofect" 1.4×10(6) Verro2GFP fibroblasts in 2 experiments. Transfected cells were plated in 20 Petri dishes (Ø=150mm) and cultured under hygromycin selection (200μgmL(-1)) for 15 days. After 12 days of drug selection, 82 resistant colonies were picked up and expanded in 24 multiwell plates for SCNT. All colonies were PCR screened and 45 (54.9%) colonies were positive. Four colonies were used in zona-free SCNT experiments with 140 Day 6 compacted morulae/blastocysts transferred into 2 synchronized sows that both became pregnant. One pregnancy went to term and delivered 5 live animals and 5 stillborn with correct hygromycin cassette integration, detected by PCR. The PCR products were sequenced in 7 animals to verify integration of promoterless targeting vector and in all 7 sequenced samples we obtained a correct insertion without any substitution/deletion. Using hygromycin selection in these experiments, we demonstrated that ZFN-mediated gene targeting can be easily done with high efficiency and is compatible with living animals. Moreover, we have validated a feasible SCNT-tested platform for further Cre-mediated site-specific gene modifications.
    Reproduction Fertility and Development 12/2013; 26(1):221-2. · 2.58 Impact Factor
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    ABSTRACT: In recent years, fetal adnexa and fluids have been recognised as important sources of mesenchymal stem cells (MSC). The aim of this study was to characterise cell populations of bovine amniotic fluid, studying phenotypic characterisation, RNA expression, and differentiation potential of samples after in vitro culture for different lengths of time following trypsinization and expansion (passage). Amniotic fluid samples were recovered at the slaughterhouse from 25 pregnant cows and harvested cells were cultured in DMEM-TCM199 (1:1) plus 10% fetal bovine serum (FBS) in 5% CO2 at 38.5°C. At passages P3 and P7, a sample for each of the 4 population found was characterised. Immunophenotypic characterisation was performed for MSC (CD90, CD105, CD44) and haematopoietic (CD14, CD34) markers by flow cytometry (FACS). Immunocytochemistry (ICC) was performed for Oct4, SSEA4, and α-SMA and the ratio between positive cells and total nuclei was evaluated. Gene expression profile was analysed by RT-PCR for pluripotency markers (Oct4, Nanog, Sox2). At the same passages chondrogenic, osteogenic and adipogenic differentiation were induced and evaluated morphologically and cytologically using, respectively, Alcian blue to identify cartilage matrix, Von Kossa for extracellular calcium deposition, and Oil Red O for intracellular lipid droplets. Cell population appeared heterogeneous and we could identify 2 main cell types: round (R) and spindle-shaped (S) cells. Each isolated sample was classified into one of the following 4 types depending on percentages of R or S cells: prevalence of S-cells (S), prevalence of R-cells (R), and samples showing both morphologies with ~10% of S-cells (S10) or 40% S-cells (S40). S-cells percentage decreased with passages in S10 and S40. After FACS, all lines were positive for CD90, CD105, CD44, and CD34 and negative for CD14 both at P3 and at P7. After ICC, Oct4 was negative in all samples analysed, few S cells stained for SSEA4 (8%) at P3 but increased at P7 to 22%; R, S10, and S40 did not express SSEA4 both at P3 and at P7. α-SMA was expressed in all samples at P3 (9.4% S; 0.9% R; 2.5% S10; 27% S40) but not at P7 (27.5% S; 0% R; 0% S10; 0% S40). After RT-PCR analyses, Oct4 was negative in all samples; at P3, Nanog was clearly positive in S-cells, weak in S40, and negative in R and S10, but all samples turned negative at P7. Sox2 was weakly expressed (S) or not expressed (S10, S40, R) at P3 and it was negative in all cells at P7. Only S showed high differentiation potential into all 3 lineages at both P3 and P7, R had the lowest differentiation potential, whereas S10 and S40 were intermediate at both end points. In conclusion, bovine amniotic fluid showed heterogeneous cell populations and S-type had the characteristics of MSCs. S10 and S40 showed more MSC markers at P3, when S cells were still present, and this aspect suggests that S population is the presumptive MSC one. Although prevalent, R-type showed only some MSC characteristics. Further studies are under way to improve S-type isolation, purification, and culture, and to determine the lifespan of these cell types.
    Reproduction Fertility and Development 12/2013; 26(1):207-8. · 2.58 Impact Factor
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    ABSTRACT: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that occurs in two clinically indistinguishable forms: sporadic (SALS) and familial (FALS), the latter linked to several gene mutations, mostly inheritable in a dominant manner. Nearly 20% of FALS forms are linked to mutations in the Cu/Zn superoxide dismutase (SOD1) gene. Research on ALS relies on transgenic models and particularly on mice carrying a glycine-to-alanine conversion at the 93rd codon (G93A) of the hSOD1 gene. Although G93A transgenic mice have been widely employed in clinical trials and basic research, doubts have been recently raised from numerous reliable sources about their suitability to faithfully reproduce human disease. Besides, the scientific community has already foreseen swine as an attractive and alternative model to nonhuman primates for modeling human diseases due to closer anatomical, physiological and biochemical features of swine rather than rodents to humans. On this basis, we have produced the first swine ALS model by in vitro transfection of cultured somatic cells combined with somatic cell nuclear transfer (SCNT). To achieve this goal we developed a SOD1(G93A) (superoxide dismutase 1 mutated in Gly93-Ala) vector, capable of promoting a high and stable transgene expression in primary porcine adult male fibroblasts (PAF). After transfection, clonal selection and transgene expression level assessment, selected SOD1(G93A) PAF colonies were used as nuclei donors in SCNT procedures. SOD1(G93A) embryos were transferred in recipient sows, and pregnancies developed to term. A total of 5 piglets survived artificial hand raising and weaning and developed normally, reaching adulthood. Preliminary analysis revealed transgene integration and hSOD1(G93A) expression in swine tissues and 360° phenotypical characterization is ongoing. We believe that our SOD1(G93A) swine would provide an essential bridge between the fundamental work done in rodent models and the reality of treating ALS. © 2013 S. Karger AG, Basel.
    Neurodegenerative Diseases 10/2013; · 3.41 Impact Factor
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    ABSTRACT: Abstract Embryologists working with livestock species were the pioneers in the field of reprogramming by somatic cell nuclear transfer (SCNT). Without the "Dolly experiment," the field of cellular reprogramming would have been slow and induced plutipotent cells (iPSCs) would not have been conceived. The major drive of the work in mammalian cloning was the interest of the breeding industry to propagate superior genotypes. Soon it was realized that the properties of oocytes could be used also to clone endangered mammalian species or to reprogram the genomes of unrelated species through what is known as interspecies (i) SCNT, using easily available oocytes of livestock species. iSCNT for cloning animals works only for species that can interbreed, and experiments with taxonomically distant species have not been successful in obtaining live births or deriving embryonic stem cell (ESC) lines to be used for regenerative medicine. There are controversial reports in the literature, but in most cases these experiments have underlined some of the cellular and molecular mechanisms that are incomplete during cell nucleus reprogramming, including the failure to organize nucleoli, silence somatic cell genes, activate the embryonic genome, and resume mitochondrial replication and function, thus indicating nucleus-cytoplasmic incompatibility.
    Cellular reprogramming. 09/2013;
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    ABSTRACT: 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), together with the maintenance of a proper level of cAMP, could improve the developmental capability of oocytes. The aim of this study was to assess: (i) dose dependent effects of cilostamide on nuclear maturation kinetics; (ii) the relationship between treatments on GJC functionality and large-scale chromatin configuration changes; (iii) and the impact of treatments on developmental competence acquisition after parthenogenic activation (PA) and SCNT. Accordingly, COC were collected from 3-6 mm antral follicles and cultured for 24 h in defined culture medium with or without 1 μM 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 up to 24 h of culture and delayed meiotic resumption as only 25.6% of cilostamide-treated oocytes reached ProMI 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.
    Biology of Reproduction 08/2013; · 4.03 Impact Factor
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    ABSTRACT: Glutaraldehyde fixation does not guarantee complete tissue biocompatibility in current clinical bioprosthetic heart valves (BHVs). Particularly, circulating anti-αGal human antibodies increase significantly from just 10 days after a BHV implantation. The inactivation of such epitope should be mandatory to meet the requirements for a perspectively safe clinical application; nevertheless, its quantitative assessment in commercially available BHVs has never been carried out. In this investigation, seven different models of BHVs were tested. The number of epitopes was determined with reference to a standard αGal source by an ELISA test. The presence of xenoantigen was subsequently confirmed by immunofluorescence analysis. Porcine tissue, knockout for the αGal epitopes, was used as negative control. Epic™ valve was the only model among those tested, in which the αGal antigen appeared to be completely shielded. Composite Trifecta™ valve exhibited conflicting results: cusps of bovine pericardial tissue were devoid of reactive αGal epitopes, while the stent cover strip of porcine pericardium still maintained 30% of active antigens originally present in native tissue. All other tested BHVs express an αGal amount not significantly different from that exhibited by porcine Mosaic(®) valve (5.2 ± 0.6 × 10(10) each 10 mg of tissue). For the first time, the quantitative evaluation of the αGal epitope in heart valve bioprostheses, already in clinical practice for about 40 yrs, was finally determined. Such quantification might provide indications of biocompatibility relevant for the selection of bioprosthetic devices and an increase in the confidence of the patient. It might become a major quality control tool in the production and redirection of future investigation in the quest for αGal-free long-lasting substitutes.
    Xenotransplantation 07/2013; · 2.57 Impact Factor
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    ABSTRACT: The aim of this study was to establish whether perturbed gene expression during cumulus oocyte development causes repeat breeding in cattle. In this study, a repeat breeder was defined as a normal estrous cycling animal that did not become pregnant after three inseminations despite the absence of clinically detectable reproductive disorders. Transcripts of genes extracted from cumulus oocyte complexes (COC) that were collected from three repeat breeder and three normally fertile Holstein Friesian heifers were compared. Up to 40 COC were collected from each heifer by means of repeated sessions of ovum pick up in the absence of hormonal stimulation; immediately plunged into liquid nitrogen; and stored at -80°C until analysis. For each heifer, RNA was extracted from the pooled COC and hybridized on GeneChip(®) Bovine Gene Array (Affymetrix). Analysis of gene expression profiles of repeat breeder and control COC showed that 178 genes were differentially expressed (log2 fold change>1.5). Of these genes, 43 (24%) were up-regulated and 135 (76%) were down-regulated in repeat breeder relative to control heifers. This altered pattern of expression occurred in genes involved in several cellular biological processes and cellular components such as metabolism, angiogenesis, substrate/ion transport, regulation/signaling, cell adhesion and cytoskeleton. From these, 13 genes potentially involved in cumulus oocyte growth were subjected to validation by qRT-PCR and nine genes (annexin A1, ANXA1; lactoferrin, LTF; interferon stimulated exonuclease 20kDa, ISG20/HEM45; oxidized low density lipoprotein receptor 1, OLR1; fatty acid desaturase 2, FADS2; glutathione S-transferase A2 and A4, GSTA2 and GSTA4; glutathione peroxidase 1, GPX1; endothelin receptor type A, EDNRA) were confirmed to be differentially expressed. This study identified potential marker genes for fertility in dairy cattle.
    Animal reproduction science 07/2013; · 1.56 Impact Factor
  • Animal Reproduction Science 07/2013; · 1.90 Impact Factor
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    ABSTRACT: 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.
    Xenotransplantation 05/2013; · 2.57 Impact Factor
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    ABSTRACT: BACKGROUND: Activation of the clotting cascade is central in acute xenograft rejection (AHXR) that occurs when pig organs are transplanted into primates. The coagulopathy reported in this model is a very complex process that involves simultaneously coagulation factors, platelets and phospholipid-bearing cells (i.e., leukocytes, red blood cells, and endothelial cells). Choosing whole blood for coagulation analysis theoretically appears more favorable compared with plasma. Whole blood rotation thromboelastometry (ROTEM(®) ) is a point-of-care global coagulation analyzer able to evaluate the characteristics of clot formation and lysis by dynamic monitoring. The aim of this study was to record thromboelastographic profiles, performed by ROTEM(®) , in a series of immunosuppressed nephrectomized primates that received a life-supporting kidney. METHODS: Of the eight primates, n = 4 received a pig kidney transgenic for human decay-accelerating factor (hDAF/Gal+); n = 2, an α 1,3-galactosyltransferase gene-knockout (GT-KO) pig kidney transgenic for human CD39, CD55, CD59 and fucosyltransferase (HTF); and n = 2, a GT-KO pig kidney transgenic for hDAF. Blood samples were collected before and at least once per week after transplantation till euthanasia. Intrinsic (INTEM) and extrinsic (EXTEM) coagulation pathways and the function of fibrinogen (FIBTEM) were evaluated. Thromboelastographic parameters considered were clotting time (CT, seconds) and clot formation time (CFT, seconds) in INTEM and EXTEM and maximum clot firmness (MCF, mm) in FIBTEM. The correlations between CT in INTEM and activated partial thromboplastin time (aPTT), CT in EXTEM and PT, CFT in INTEM and EXTEM, and platelet counts and MCF in FIBTEM and fibrinogen plasma levels were also considered. RESULTS: In all animals, thromboelastographic profiles showed progressive prolongation of CT (activation of coagulative cascade) in INTEM. A close correspondence was observed between (i) the prolongation of the CFT values (propagation of clot formation), both in INTEM and EXTEM, and the decrease in platelet counts; (ii) the reduction in MCF values (clot firmness) in FIBTEM and the decrease in fibrinogen plasma levels. No concordance between CT in INTEM and aPTT and between CT in EXTEM and PT was observed. CONCLUSIONS: Our study demonstrated that ROTEM(®) analyzer could be a useful and complementary tool to study the consumptive coagulopathy, either "compensated" or "non-compensated," that takes place when transgenic pig kidneys are transplanted into primates. Larger and prospective studies are needed to confirm our results and to evaluate the role of ROTEM(®) to guide the management of consumptive coagulopathy in order to prolong the survival of the transplanted organ.
    Xenotransplantation 02/2013; · 2.57 Impact Factor
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    ABSTRACT: Design of new strategies to improve in vitro embryo production (IVP) efficiency, such as pre-maturation culture with cilostamide (CILO PMC) aimed to prolong oocytes-cumulus cells gap junction-mediated communications (GJC) functionality while delaying meiotic resumption, should consider the high heterogeneity of cumulus-oocyte complexes (COCs) retrieved from ovarian follicles. In the mare, main factors contributing to this heterogeneity are: the follicle diameter, the cumulus oophorus morphology and the reproductive seasonality. Thus, the objectives of this study were: 1) to identify a population of equine oocytes that would benefit from a CILO PMC, based on the assessment of the GJC functionality by Lucifer Yellow microinjection, the oocyte chromatin configuration by DNA staining, and the meiotic competence after in vitro maturation (IVM), in COCs of different origins, since these parameters are indicative of the oocyte metabolic state and 2) to assess the effect of CILO PMC, in COCs of different origins, on the GJC functionality, the meiotic and embryonic developmental competence after IVM and intracytoplasmic sperm injection (ICSI). COCs with Compact (Cp) or Expanded (Ex) cumulus oophorus were collected from <1, 1-2 and >2 cm follicles during winter, spring-summer and fall, and cultured with our standard IVM protocol (CTRL) or prematurated in the same medium with 10-4 UI r-hFSH/ml and 10 µM Cilostamide (CILO PMC) for different times. When meiotic and developmental competences were assessed, COCs were subjected to CILO PMC for 6 h and than to standard IVM and ICSI. Data were analyzed by Chi square test. Overall, our results suggest that COCs that would more probably benefit from CILO PMC are those with Cp cumuli from <1 and 1-2 cm follicles collected during spring-summer and fall. In fact, they have a lower meiotic competence after 24h IVM (P<0.05), with a mean MII rate of 40%, compared to all the other groups, in which MII rate is around 60%. Moreover, they show a higher % (P<0.05) of COC with open GJC (70%), when compared with both Cp COC in the winter season and Exp COC from all follicular classes in all seasons tested, in which more that 50% of COCs have closed GJC. Independently from the season, chromatin configuration analysis reveals that oocytes with Cp cumuli from 1-2 cm follicles are in a more advanced stage of differentiation since their chromatin is more condensed (P<0.05). Study on GJC indicates that functionality can be maintained up to 10h in Cp COCs from 1-2 cm using CILO PMC when compared to the CTRL group, in which a major GJC drop is detected at 10h. In fact, COCs with open GJC are 74% and 44% in CILO PMC and CTRL groups respectively at 10 h (P<0.05) On the contrary, the exact timing for the treatment of Cp COCs from <1 cm follicles remains to be determined since GJC functionality drops after 16h in the CTRL group, with only 14% of COCs with open GJC, and no differences are observed between groups. Finally, initial evaluation of the effect of CILO PMC for 6h indicates that it does not affect the rate of embryos obtained after ICSI. Evaluation of embryo quality is in progress to assess whether CILO PMC could improve IVP efficiency beyond embryo yield. In conclusion, our data could be useful in setting up IVM strategies to improve horse IVP efficiency as well as the understanding of horse oocyte biology. Funding: Grant n. 26096200 "Ex Ovo Omnia" Regione Sardegna-Lombardia and ‘Dote Ricerca’ FSE, Regione Lombardia.
    46th Annual Meeting of the Society for the Study of Reproduction; 01/2013
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    ABSTRACT: Induction of neural differentiation from embryonic pluripotent stem cells (ES/EpiSc), both of mouse and primates, has been extensively published by several research teams. However, direct derivation of organized neuroectoderm in vitro from blastocyst stage embryos of rodents or primates has not been reported so far. Here we describe a method of direct neural differentiation from the inner cell mass cells of preimplantation bovine embryos, without the intermediate step of ES/EpiSc cells derivation (Lazzari et al., Stem cells 24:2514-2521, 2006). Proliferating neural precursors cells lines, and both central and peripheral nervous system derivatives, can be obtained providing a unique in vitro model of early neurulation events in mammals.
    Methods in molecular biology (Clifton, N.J.) 01/2013; 1074:199-207. · 1.29 Impact Factor

Publication Stats

3k Citations
426.11 Total Impact Points

Institutions

  • 2005–2014
    • University of Bologna
      • Department of Veterinary Medical Sciences DIMEVET
      Bolonia, Emilia-Romagna, Italy
    • University of Milan
      Milano, Lombardy, Italy
  • 2003–2013
    • Istituto Sperimentale Italiano Lazzaro Spallanzani
      Rivolta, Lombardy, Italy
  • 2009–2012
    • AVANTEA srl
      Cremona, Lombardy, Italy
    • Massachusetts General Hospital
      Boston, Massachusetts, United States
  • 2008
    • San Raffaele Scientific Institute
      Milano, Lombardy, Italy
  • 2004–2008
    • Universiteit Utrecht
      • Gezondheidszorg Paard (DGP)
      Utrecht, Provincie Utrecht, Netherlands
    • Catholic University of Louvain
      • Institute of Life Sciences
      Louvain-la-Neuve, WAL, Belgium
    • French National Institute for Agricultural Research
      • Biologie du Développement et Reproduction (BDR)
      Paris, Ile-de-France, France
  • 1994–2008
    • Laboratorio di Tecnologie Oncologiche (LATO)
      Cefalù, Sicily, Italy
    • Babraham Institute
      Cambridge, England, United Kingdom
  • 1999
    • Netherlands Institute for Space Research, Utrecht
      Utrecht, Utrecht, Netherlands