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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 (

The present invention concerns the field of gene integration and durable gene expression in specific genomic sites of transgenic animals suitable for the generation of tools for biomedical research and biotechnological applications.

In recent years fetal adnexa and fluids have been recognized as important sources of mesenchymal stem cells (MSCs). The aim of this study was to characterize cell populations of bovine amniotic fluid studying phenotypic characterization, RNA expression and differentiation potential of samples after in vitro culture for different length of time following trypsinization and expansion (passage). Amniotic fluid samples were recovered at slaughterhouse from 25 pregnant cows and harvested cells were cultured in DMEM-TCM199 (1:1) plus 10% FBS in 5%CO2 at 38.5 °C. At passages P3 and P7, a sample for each of the 4 population found was characterized. Immunophenotypic characterization was performed for MSC (CD90, CD105, CD44) and hematopoietic (CD14, CD34) markers by flow cytometry (FACS). Immunocytochemistry (ICC) was performed for Oct4, SSEA4 and α-SMA and ratio between positive cells and total nuclei was evaluated. Gene expression profile was analyzed 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 glycosaminoglycans of 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 about 10% of S-cells (S10) or 40% S-cells (S40). S-cell 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-type 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-cells showed high differentiation potential into all 3 lineages at both P3 and P7, R-cells had the lowest differentiation potential while 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 population was 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, culture and to determine the lifespan of these cell types. Work supported by grant PRIN2009.

The cloning of equines by somatic cell nuclear trans- fer (SCNT) was first reported in 2003 (Galli et al., 2003; Woods et al., 2003), 6 years after the birth of Dolly the sheep (Wilmut et al., 1997). Amongst the mammalian spe- cies that have been cloned (Campbell et al., 2007), equines are among the last. In general, the application of assisted reproduction technologies in equines has lagged behind that of other livestock species (Galli et al., 2007) in the poor interest expressed by the industry (still today, for example, the thoroughbred does not allow artificial insemi- nation) and also the limited resources devoted to this kind of research and the few laboratories working in this area. In fact the first equine clone, a mule (Woods et al., 2003), was obtained from in vivo matured oocytes that were transferred back to the oviduct of a synchronized recipi- ent mare immediately after nuclear transfer and activation. This approach was successful because it limited to a mini- mum the time exposure to the in vitro environment. This approach of using in vivo matured oocytes was, however, not sustainable from a practical point of view. In contrast, the first cloned horses (Galli et al., 2003; Lagutina et al., 2005) and those that followed later (Hinrichs et al., 2006, 2007) were all derived from abattoir-recovered and in vitro matured oocytes that, following enucleation and embryo reconstruction, were cultured in vitro to the blastocyst stage prior to non-surgical embryo transfer to a recipient mare. This method could be applied only after the develop- ment of the procedures for in vitro maturation of oocytes, fertilization by ICSI, and in vitro culture of embryos capable of establishing normal pregnancies and generat- ing offspring (Galli et al., 2007; Hinrichs, 2010). In addi- tion to the refinement of the in vitro protocols for oocyte maturation and embryo culture, SCNT protocols had to be adapted and optimized for the equine, which, because of its anatomical and physiological features, provides a lim- ited number of oocytes per ovary when compared to cat- tle or pigs. The protocols currently applied for nuclear transfer are based on a zona-free system (Lagutina et al., 2007) to enhance cell fusion or a piezo-electric device (Westhusin et al., 2003) for enucleation and microinjec- tion of the nucleus directly into the oocyte. Reconstructed embryos also require a specific activation protocol (Hinrichs et al., 2006; Lazzari et al., 2002).

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 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 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.

Introduction - Amniotic fluid (AF) is a reservoir 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 bovine MSCs from AF (bAF-MSCs) as alternative sources of primitive multipotent stem cells in a species that could be an attractive large-animal model for biomedical and biotechnology researches. Materials and Methods - Samples were recovered, at slaughterhouse, from 18 pregnant cows at different gestational ages established on crown-rump length (1: 0-105 d; 2: 106-160 d; 3: 161-200 d; 4: 201-240 d; 5: 241 d-term of pregnancy). Cells were isolated by centrifugation and cultured in DMEM-TCM 199 (1:1) plus 10% FBS. At passage (P) 4, chondrogenic, osteogenic and adipogenic differentiation were induced (n=16) and evaluated morphologically and cytologically (Alcian Blue, Von Kossa and Oil Red O stainings respectively). Phenotypic characterization for stem cell markers was performed at P3 or P4 by flow cytometry for CD105, CD90, CD45 and CD14 (n=16), by immunocytochemistry (ICC) for Oct4, SSEA1, SSEA4 and α-SMA (n=4) and by RT-PCR analysis for Oct4, Nanog and Sox2 (n=7). In both ICC and RT-PCR, bovine fibroblasts were used as negative control. Results – Mean collected AF volume was 35 ml and 200-3636 cells/ml were recovered. Cells were isolated from 16/18 samples. At P0, cell populations appeared heterogeneous and we could identify 4 different cell types: round, spindle-shaped, fibroblastoid and large-flat cells. These different populations were observed at every gestational age analyzed. Through passages, only round cells (n=15/16) predominated. In only one sample, spindle-shaped cells prevailed. After 30 d of culture mean cell doublings were 14,1±3,2 and mean doubling time was 57,7±27,9 hrs without differences related to gestational age or sample (p>0.05). Cells were able to differentiate into osteocytes and adipocytes; chondrogenesis was clearly obtained only from samples recovered within 140 d of pregnancy (n=9). Percentage of cells positive for CD90 was 73,0±17,2, for CD105 45,8±35,7, for CD14 5,6±11,2 and for CD45 13,4±11,3; no differences were observed among different gestational ages (p>0,05). ICC demonstrated low presence of SSEA4 positive cells (0,05%) in 1 sample, the presence of α-SMA in all 4 samples (mean 11,8±14.1%, range 2.5-32.4%) and the lack of Oct4 and SSEA1. RT-PCR analysis, performed at P4, showed expression of Nanog in 1/7 sample and weak expression in 4/7 samples. Low expression of SOX2 was detected in 2/7 samples. Conclusions - Based on these preliminary results, in the bovine AF there is an heterogeneous cell population containing also multipotent MSCs, identified by their differentiation ability and phenotypic characteristics typical of MSCs. Unlike human AF-MSCs, only a fraction of bAF-MSCs are positive for some pluripotency markers. Current work is ongoing to further characterize other cell phenotypes selected after long term culture.

Foetal adnexa contain a heterogeneous mixture of cells including a stem cell population that express a panel of pluripotency markers, such as Oct4, Nanog, Sox2 and SSEA4 (Pappa and Anagnou, 2009; Dyvia et al, 2012). These extra-embryonic tissues and fluids represent viable, accessible and safe sources. The aim of this work was to evaluate pluripotency markers expression in mesenchymal stem cells isolated from equine amniotic fluid (eAF-MSCs), Wharton’s jelly (eWJ-MSCs) and umbilical cord blood (eUCB-MSCs) as continuation of the characterization of these cells previously performed by our research group (Iacono et al., 2012). Equine AF, WJ and UCB samples were recovered at parturition from 3 mares and (46.7 ± 14.4) ml of AF, (2.4 ± 2.2) g of WJ and (23.3 ± 14.4) ml of UBC were collected at foaling. Isolated cells were isolated and cultured as previously described (Iacono et al. 2012). Briefly, cells were cultured in DMEM-TCM 199 (1:1) plus 10% FBS and incubated in a 5% CO2 humidified atmosphere at 38.5 °C. At passage 3 characterization was performed by immunocytochemistry (ICC) and RT-PCR. ICC was carried out for SSEA4 protein fixing cells with paraformaldehyde (4%), doing an overnight incubation at 4°C with anti-SSEA4 primary antibody and then incubating cells with FITC-labeled secondary antibody for 1 hr at room temperature in the dark. Nuclei were stained with Hoechst and the percentages of positive cells were obtained doing ratio between FITC+ cells and nuclei. Equine skin fibroblasts were used as negative control. RT-PCR was performed to analyze mRNA expression of Nanog and Sox2. Equine blastocysts and equine skin fibroblasts were used respectively as positive and negative controls and GAPDH was employed as reference gene. ICC analysis showed expression of the embryonic stem cells antigen SSEA4 in eAF-MSCs (14.8 ± 9.5 %), eWJ-MSCs (8.9 ± 2.6 %) and eUCB-MSCs (7.1 ± 4.8 %). On the other hand, expression of Nanog and Sox2 was not found in all samples, unlike human AF-MSCs and WJ-MSCs. ICC and RT-PCR analyses for the embryonic marker Oct4 (data not reported) gave unclear results so further studies are necessary. Results obtained in the present study reveal that equine MSCs derived from foetal adnexa are an heterogeneous population and express low level of the embryonic marker SSEA4, but do not express embryonic pluripotent markers like Sox2 and Nanog as described in the literature for other species. So these cells could represent an intermediate stage between pluripotent embryonic stem cells and lineage-restricted adult stem cells, as previously demonstrated for human MSCs (De Coppi et al. 2007; Pappa and Anagnou, 2009). Further work is ongoing to find specific primers and antibodies to assess the expression of OCT4.

Equine Assisted Reproduction and Embryo Technologies

The present invention concerns a novel transgenic animal as model of Amyotrophic Lateral Sclerosis (ALS).