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1. In Vitro-Produced Equine Blastocysts Exhibit Greater Dispersal and Intermingling of Inner Cell Mass Cells than In Vivo Embryos

Author

Umair, Muhammad, Scheeren, Veronica Flores da Cunha, Beitsma, Mabel M, Colleoni, Silvia, Galli, Cesare, Lazzari, Giovanna, de Ruijter-Villani, Marta, Stout, Tom A E, Claes, Anthony, Voortplanting paard, and CS_Fertility

Subjects
blastocysts, in vitro embryo production, Inorganic Chemistry, cell lineage segregation, Organic Chemistry, Physical and Theoretical Chemistry, Molecular Biology, ICM, Spectroscopy, Catalysis, equine, Computer Science Applications
Abstract

In vitro production (IVP) of equine embryos is increasingly popular in clinical practice but suffers from higher incidences of early embryonic loss and monozygotic twin development than transfer of in vivo derived (IVD) embryos. Early embryo development is classically characterized by two cell fate decisions: (1) first, trophectoderm (TE) cells differentiate from inner cell mass (ICM); (2) second, the ICM segregates into epiblast (EPI) and primitive endoderm (PE). This study examined the influence of embryo type (IVD versus IVP), developmental stage or speed, and culture environment (in vitro versus in vivo) on the expression of the cell lineage markers, CDX-2 (TE), SOX-2 (EPI) and GATA-6 (PE). The numbers and distribution of cells expressing the three lineage markers were evaluated in day 7 IVD early blastocysts ( n = 3) and blastocysts ( n = 3), and in IVP embryos first identified as blastocysts after 7 (fast development, n = 5) or 9 (slow development, n = 9) days. Furthermore, day 7 IVP blastocysts were examined after additional culture for 2 days either in vitro ( n = 5) or in vivo (after transfer into recipient mares, n = 3). In IVD early blastocysts, SOX-2 positive cells were encircled by GATA-6 positive cells in the ICM, with SOX-2 co-expression in some presumed PE cells. In IVD blastocysts, SOX-2 expression was exclusive to the compacted presumptive EPI, while GATA-6 and CDX-2 expression were consistent with PE and TE specification, respectively. In IVP blastocysts, SOX-2 and GATA-6 positive cells were intermingled and relatively dispersed, and co-expression of SOX-2 or GATA-6 was evident in some CDX-2 positive TE cells. IVP blastocysts had lower TE and total cell numbers than IVD blastocysts and displayed larger mean inter-EPI cell distances; these features were more pronounced in slower-developing IVP blastocysts. Transferring IVP blastocysts into recipient mares led to the compaction of SOX-2 positive cells into a presumptive EPI, whereas extended in vitro culture did not. In conclusion, IVP equine embryos have a poorly compacted ICM with intermingled EPI and PE cells; features accentuated in slowly developing embryos but remedied by transfer to a recipient mare.

Published
2023

2. Oxidative Stress in Structural Valve Deterioration: A Longitudinal Clinical Study

Author

Galiñanes, Manuel, Casós, Kelly, Blasco-Lucas, Arnau, Permanyer, Eduard, Máñez, Rafael, Le Tourneau, Thierry, Barquinero, Jordi, Schwartz, Simon, Bottio, Tomaso, Roussel, Jean Christian, Fellah-Hebia, Imen, Sénage, Thomas, Evangelista Masip, Arturo, Badano, Luigi P., Ruiz-Majoral, Alejandro, Galli, Cesare, Padler-Karavani, Vered, Soulillou, Jean-Paul, Vidal Guitart, Xavier, Cozzi, Emanuele, Costa, Cristina, Universitat Autònoma de Barcelona, Universitat Autònoma de Barcelona (UAB), Vall d'Hebron University Hospital [Barcelona], Vall d’Hebron Research Institute (VHIR), Llobregat Hospital [Barcelona], Quironsalud Teknon Heart Institute [Barcelona, Spain] (QTHI), Institut d'Investigació Biomèdica de Bellvitge [Barcelone] (IDIBELL), Bellvitge University Hospital [Barcelona, Spain], Centre d'investigation clinique (CIC) de Nantes -CIC Plurithématique (CIC 0004 - Nantes), Direction Générale de l'Organisation des Soins (DGOS)-Institut National de la Santé et de la Recherche Médicale (INSERM), unité de recherche de l'institut du thorax UMR1087 UMR6291 (ITX), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Nantes Université - UFR de Médecine et des Techniques Médicales (Nantes Univ - UFR MEDECINE), Nantes Université - pôle Santé, Nantes Université (Nantes Univ)-Nantes Université (Nantes Univ)-Nantes Université - pôle Santé, Nantes Université (Nantes Univ)-Nantes Université (Nantes Univ), Università degli Studi di Padova = University of Padua (Unipd), Azienda Ospedale Università di Padova = Hospital-University of Padua (AOUP), Centre hospitalier universitaire de Nantes (CHU Nantes), Institut du Thorax [Nantes], Università degli Studi di Milano-Bicocca = University of Milano-Bicocca (UNIMIB), Istituti di Ricovero e Cura a Carattere Scientifico (IRCCS), Fondazione Avantea [Cremona, Italy], Tel Aviv University (TAU), Centre de Recherche en Transplantation et Immunologie - Center for Research in Transplantation and Translational Immunology (U1064 Inserm - CR2TI), Institut National de la Santé et de la Recherche Médicale (INSERM)-Nantes Université - UFR de Médecine et des Techniques Médicales (Nantes Univ - UFR MEDECINE), Institut Català de la Salut [Barcelona, Spain] (ICS), L’Hospitalet de Llobregat [Barcelona, Spain], The project was funded by the European Commission (FP7/2007–2013, Grant Agreement 603049: accessed on 1 September 2013, and PERIS SLT002/16/00445 funded by the Department of Health of the Generalitat de Catalunya, both granted to C.C., and cofunded by FEDER funds, a way to build Europe. IDIBELL benefits from the support of CERCA., European Project: 603049,EC:FP7:HEALTH,FP7-HEALTH-2013-INNOVATION-1,TRANSLINK(2013), Centre d’Investigation Clinique de Nantes (CIC Nantes), Université de Nantes (UN)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre hospitalier universitaire de Nantes (CHU Nantes), KERANDEL-DION, Céline, Defining the role of xeno-directed and autoimmune events in patients receiving animal-derived bioprosthetic heart valves - TRANSLINK - - EC:FP7:HEALTH2013-09-01 - 2017-08-31 - 603049 - VALID, Galinanes, M, Casos, K, Blasco-Lucas, A, Permanyer, E, Manez, R, Le Tourneau, T, Barquinero, J, Schwartz, S, Roussel, T, Fellah-Hebia, I, Senage, T, Evangelista, A, Badano, L, Ruiz-Majoral, A, Galli, C, Padler-Karavani, V, Soulillou, J, Vidal, X, Cozzi, E, and Costa, C

Subjects
Estrès oxidatiu, [SDV]Life Sciences [q-bio], Structural valve deterioration, Lipid peroxidation, Prosthesis Design, Biochemistry, structural valve deterioration, aortic valve, biological heart valves, lipid peroxidation, oxidative stress, protein nitration, transcatheter aortic valve implantation, Humans, Aortic valve, Molecular Biology, Heart valve prosthesis, Heart valves, oxidative stre, Transcatheter aortic valve implantation, Pròtesis valvulars cardíaques, MED/11 - MALATTIE DELL'APPARATO CARDIOVASCOLARE, Aortic Valve Stenosis, Vàlvules cardíaques, biological heart valve, Prosthesis Failure, [SDV] Life Sciences [q-bio], Treatment Outcome, Oxidative stress, Heart Valve Prosthesis, Biological heart valves, Protein nitration
Abstract

International audience; The cause of structural valve deterioration (SVD) is unclear. Therefore, we investigated oxidative stress markers in sera from patients with bioprosthetic heart valves (BHVs) and their association with SVD. Blood samples were taken from SVD (Phase A) and BHV patients during the first 24 (Phase B1) and >48 months (Phase B2) after BHV implantation to assess total antioxidant capacity (TAC), malondialdehyde (MDA), and nitrotyrosine (NT). The results show that MDA levels increased significantly 1 month after surgery in all groups but were higher at 6 months only in incipient SVD patients. NT levels increased gradually for the first 24 months after implantation in the BHV group. Patients with transcatheter aortic valve implantation (TAVI) showed even higher levels of stress markers. After >48 months, MDA and NT continued to increase in BHV patients with a further elevation after 60–72 months; however, these levels were significantly lower in the incipient and established SVD groups. In conclusion, oxidative stress may play a significant role in SVD, increasing early after BHV implantation, especially in TAVI cases, and also after 48 months’ follow-up, but decreasing when SVD develops. Oxidative stress potentially represents a target of therapeutic intervention and a biomarker of BHV dysfunction.

Published
2022
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3. The ART of bringing extinction to a freeze e History and future of species conservation, exemplified by rhinos

Author

Hildebrandt, Thomas B., Hermes, Robert, Goeritz, Frank, Appeltant, Ruth, Colleoni, Silvia, de Mori, Barbara, Diecke, Sebastian, Drukker, Micha, Galli, Cesare, Hayashi Katzuiko, Lazzari, Giovanna, Loi, Pasqualino, Payne, J, Renfree, Marilyn, Seet, Steven, Stejskal, Jan, Swegen, A, Williams, Suzannah A., Zainuddin, Zainal Zahari, and Holtze, Susanne

Abstract

The ongoing mass extinction of animal species at an unprecedented rate is largely caused by human activities. Progressive habitat destruction and fragmentation is resulting in accelerated loss of biodiversity on a global scale. Over decades, captive breeding programs of non-domestic species were characterized by efforts to optimize species-specific husbandry, to increase studbook-based animal exchange, and to improve enclosure designs. To counter the ongoing dramatic loss of biodiversity, new approaches are warranted. Recently, new ideas, particularly the application of assisted reproduction technologies (ART), have been incorporated into classical zoo breeding programs. These technologies include semen and oocyte collection, artificial insemination, and in-vitro embryo generation. More futuristic ideas of advanced ART (aART) implement recent advances in biotechnology and stem-cell related approaches such as cloning, inner cell mass transfer (ICM), and the stem-cell-associated techniques (SCAT) for the generation of gametes and ultimately embryos of highly endangered species, such as the northern white rhinoceros (Ceratotherium simum cottoni) of which only two female individuals are left. Both, ART and aART greatly depend on and benefit from the rapidly evolving cryopreservation techniques and biobanking not only of genetic, but also of viable cellular materials suitable for the generation of induced pluripotent stem cells (iPSC). The availability of cryopreserved materials bridges gaps in time and space, thereby optimizing the available genetic variability and enhancing the chance to restore viable populations.

Published
2021
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4. The role of antibody responses against glycans in bioprosthetic heart valve calcification and deterioration

Author

Thomas Senage, Anu Paul, Thierry Le Tourneau, Imen Fellah-Hebia, Marta Vadori, Salam Bashir, Manuel Galiñanes, Tomaso Bottio, Gino Gerosa, Arturo Evangelista, Luigi P. Badano, Alberto Nassi, Cristina Costa, Galli Cesare, Rizwan A. Manji, Caroline Cueff de Monchy, Nicolas Piriou, Romain Capoulade, Jean-Michel Serfaty, Guillaume Guimbretière, Etienne Dantan, Alejandro Ruiz-Majoral, Guénola Coste du Fou, Shani Leviatan Ben-Arye, Liana Govani, Sharon Yehuda, Shirley Bachar Abramovitch, Ron Amon, Eliran Moshe Reuven, Yafit Atiya-Nasagi, Hai Yu, Laura Iop, Kelly Casós, Sebastián G. Kuguel, Arnau Blasco-Lucas, Eduard Permanyer, Fabrizio Sbraga, Roger Llatjós, Gabriel Moreno-Gonzalez, Melchor Sánchez-Martínez, Michael E. Breimer, Jan Holgersson, Susann Teneberg, Marta Pascual-Gilabert, Alfons Nonell-Canals, Yasuhiro Takeuchi, Xi Chen, Rafael Mañez, Jean-Christian Roussel, Jean-Paul Soulillou, Emanuele Cozzi, Vered Padler-Karavani, unité de recherche de l'institut du thorax UMR1087 UMR6291 (ITX), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Nantes Université - UFR de Médecine et des Techniques Médicales (Nantes Univ - UFR MEDECINE), Nantes Université - pôle Santé, Nantes Université (Nantes Univ)-Nantes Université (Nantes Univ)-Nantes Université - pôle Santé, Nantes Université (Nantes Univ)-Nantes Université (Nantes Univ), MethodS in Patients-centered outcomes and HEalth ResEarch (SPHERE), Université de Tours (UT)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Nantes Université - UFR des Sciences Pharmaceutiques et Biologiques (Nantes Université - UFR Pharmacie), Tel Aviv University (TAU), Brigham & Women’s Hospital [Boston] (BWH), Harvard Medical School [Boston] (HMS), Università degli Studi di Padova = University of Padua (Unipd), Universitat Autònoma de Barcelona (UAB), Vall d'Hebron University Hospital [Barcelona], Università degli Studi di Milano-Bicocca = University of Milano-Bicocca (UNIMIB), Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Llobregat Hospital [Barcelona], Fondazione Avantea [Cremona, Italy], St. Boniface Hospital Albrechtsen Research Centre [Winnipeg], Israel Institute for Biological Research (IIBR), University of California [Davis] (UC Davis), University of California (UC), Institut d'Investigació Biomèdica de Bellvitge [Barcelone] (IDIBELL), Quironsalud Teknon Heart Institute [Barcelona, Spain] (QTHI), Mind the Byte [Barcelona, Spain], Molomics [Barcelona, Spain], Sahlgrenska Academy at University of Gothenburg [Göteborg], Institut Universitari de Ciència i Tecnologia [Barcelona, Spain] ((Inkemia Group) IUCT), DevsHealth [Barcelona, Spain] (DH), University College of London [London] (UCL), Bellvitge University Hospital [Barcelona, Spain], Centre de Recherche en Transplantation et Immunologie - Center for Research in Transplantation and Translational Immunology (U1064 Inserm - CR2TI), Institut National de la Santé et de la Recherche Médicale (INSERM)-Nantes Université - UFR de Médecine et des Techniques Médicales (Nantes Univ - UFR MEDECINE), Dantan, Etienne, Senage, T, Paul, A, Le Tourneau, T, Fellah-Hebia, I, Vadori, M, Bashir, S, Galinanes, M, Bottio, T, Gerosa, G, Evangelista, A, Badano, L, Nassi, A, Costa, C, Cesare, G, Manji, R, Cueff de Monchy, C, Piriou, N, Capoulade, R, Serfaty, J, Guimbretiere, G, Dantan, E, Ruiz-Majoral, A, Coste du Fou, G, Leviatan Ben-Arye, S, Govani, L, Yehuda, S, Bachar Abramovitch, S, Amon, R, Reuven, E, Atiya-Nasagi, Y, Yu, H, Iop, L, Casos, K, Kuguel, S, Blasco-Lucas, A, Permanyer, E, Sbraga, F, Llatjos, R, Moreno-Gonzalez, G, Sanchez-Martinez, M, Breimer, M, Holgersson, J, Teneberg, S, Pascual-Gilabert, M, Nonell-Canals, A, Takeuchi, Y, Chen, X, Manez, R, Roussel, J, Soulillou, J, Cozzi, E, Padler-Karavani, V, Institut Català de la Salut, [Senage T] Institut du Thorax, Institut National de la Santé et de la Recherche Médicale UMR1087, University Hospital, Nantes, France. Institut National de la Santé et de la Recherche Médicale UMR 1246-SPHERE, Nantes University, Tours University, Nantes, France. [Paul A] Department of Cell Research and Immunology, Shmunis School of Biomedicine and Cancer Research, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel. Department of Pathology, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA. [Le Tourneau T, Fellah-Hebia I] Institut du Thorax, Institut National de la Santé et de la Recherche Médicale UMR1087, University Hospital, Nantes, France. [Vadori M] Consortium for Research in Organ Transplantation, Ospedale Giustinianeo, Padova, Italy. [Bashir S] Department of Cell Research and Immunology, Shmunis School of Biomedicine and Cancer Research, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel. [Galiñanes M] Servei de Cirurgia Cardíaca, Vall d’Hebron Institut de Recerca (VHIR), Barcelona, Spain. Vall d’Hebron Hospital Universitari, Barcelona, Spain. Universitat Autònoma de Barcelona, Bellaterra, Spain. [Evangelista A] Servei de Cardiologia, Vall d’Hebron Institut de Recerca (VHIR), Barcelona, Spain. Vall d’Hebron Hospital Universitari, Barcelona, Spain. [Casós K] Servei de Cirurgia Cardíaca, Vall d’Hebron Institut de Recerca (VHIR), Barcelona, Spain. Vall d’Hebron Hospital Universitari, Barcelona, Spain. Universitat Autònoma de Barcelona, Bellaterra, Spain. Infectious Diseases and Transplantation Division, Institut d’Investigació Biomèdica de Bellvitge, L’Hospitalet de Llobregat, Barcelona, Spain. Grup de Recerca en Malalties Cardiovasculars, Vall d’Hebron Institut de Recerca (VHIR), Barcelona, Spain. Vall d’Hebron Hospital Universitari, Barcelona, Spain. Universitat Autònoma de Barcelona, Bellaterra, Spain. [Blasco-Lucas A] Servei de Cirurgia Cardíaca, Vall d’Hebron Institut de Recerca (VHIR), Barcelona, Spain. Vall d’Hebron Hospital Universitari, Barcelona, Spain. Universitat Autònoma de Barcelona, Bellaterra, Spain. Cardiac Surgery Department, Bellvitge University Hospital, L’Hospitalet de Llobregat, Barcelona, Spain. [Permanyer E] Servei de Cirurgia Cardíaca, Vall d’Hebron Institut de Recerca (VHIR), Barcelona, Spain. Vall d’Hebron Hospital Universitari, Barcelona, Spain. Universitat Autònoma de Barcelona, Bellaterra, Spain. Department of Cardiac Surgery, Quironsalud Teknon Heart Institute, Barcelona, Spain, and Vall d'Hebron Barcelona Hospital Campus

Subjects
[SDV]Life Sciences [q-bio], Immunology, enfermedades cardiovasculares::enfermedades cardíacas [ENFERMEDADES], Cardiovascular, Medical and Health Sciences, General Biochemistry, Genetics and Molecular Biology, Mice, Polysaccharides, Animals, Humans, Prospective Studies, Polysaccharide, Bioprosthesi, Bioprosthesis, 5.3 Medical devices, Animal, Calcinosis, Galactose, Otros calificadores::Otros calificadores::/cirugía [Otros calificadores], MED/11 - MALATTIE DELL'APPARATO CARDIOVASCOLARE, General Medicine, Aortic Valve Stenosis, Aortic Valve Stenosi, Other subheadings::Other subheadings::/surgery [Other subheadings], Vàlvula aòrtica - Cirurgia, [SDV] Life Sciences [q-bio], Cardiovascular Diseases::Heart Diseases [DISEASES], Prospective Studie, Heart Disease, Aortic Valve, Immunoglobulin G, Antibody Formation, Calcinosi, Development of treatments and therapeutic interventions, Immunoglobulines, Human
Abstract

Outcomes research; Risk factors Investigación de resultados; Factores de riesgo Recerca dels resultats; Factors de risc Bioprosthetic heart valves (BHVs) are commonly used to replace severely diseased heart valves but their susceptibility to structural valve degeneration (SVD) limits their use in young patients. We hypothesized that antibodies against immunogenic glycans present on BHVs, particularly antibodies against the xenoantigens galactose-α1,3-galactose (αGal) and N-glycolylneuraminic acid (Neu5Gc), could mediate their deterioration through calcification. We established a large longitudinal prospective international cohort of patients (n = 1668, 34 ± 43 months of follow-up (0.1–182); 4,998 blood samples) to investigate the hemodynamics and immune responses associated with BHVs up to 15 years after aortic valve replacement. Early signs of SVD appeared in

Published
2021
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5. Can We Extrapolate From a Cmah -/- Ldlr -/- Mouse Model a Susceptibility for Atherosclerosis in Humans?

Author

Soulillou, Jean-Paul, Cozzi, Emanuele, Galli, Cesare, Bach, Jean-Marie, Centre de Recherche en Transplantation et Immunologie (U1064 Inserm - CRTI), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN), Institut de transplantation urologie-néphrologie (ITUN), Université de Nantes (UN)-Centre hospitalier universitaire de Nantes (CHU Nantes), University Hospital of Padua, Avantea Laboratory of Reproductive Technologies [Cremona, Italy], Immuno-Endocrinologie Cellulaire et Moléculaire (IECM), Institut National de la Recherche Agronomique (INRA)-Université de Nantes (UN)-Ecole Nationale Vétérinaire de Nantes, Université de Nantes (UN)-Ecole Nationale Vétérinaire de Nantes-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Le Bihan, Sylvie, Azienda Ospedale Università di Padova = Hospital-University of Padua (AOUP), and Université de Nantes (UN)-Ecole Nationale Vétérinaire de Nantes-École nationale vétérinaire, agroalimentaire et de l'alimentation Nantes-Atlantique (ONIRIS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)

Subjects
[SDV] Life Sciences [q-bio], [SDV.MHEP] Life Sciences [q-bio]/Human health and pathology, [SDV]Life Sciences [q-bio], Models, Animal, Animals, Humans, Neuraminic Acids, Letters, Atherosclerosis, [SDV.MHEP]Life Sciences [q-bio]/Human health and pathology, ComputingMilieux_MISCELLANEOUS, Mixed Function Oxygenases
Abstract

International audience

Published
2020
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6. Food and Intellectual Property Rights: a comprehensive approach

Author

Galli, Cesare

Abstract

Il settore agroalimentare è oggetto di speciale attenzione da parte del diritto IP. Da un lato la protezione dei segni che indicano l'origine geografica dei prodotti, pur non essendo ancora del tutto armonizzata a livello internazionale, sta mostrando significative convergenze, basate sulla tutela della reputazione dei prodotti e contro l'inganno del pubblico. Dall'altro lato anche le innovazioni in questo campo hanno trovato nuove forme di protezione, dai brevetti sugli OGM e dai brevetti speciali per le nuove varietà vegetali alle varie forme di protezione del food design e persino del food lighting. Il denominatore comune di queste regole è un approccio “realistico”, che commisura la tutela delle diverse creazioni al valore che esse presentano in concreto sul mercato e nel “mondo della vita”., Cibo e diritto. Una prospettiva comparata. Volume 2

Published
2020
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7. Generation of fertile cloned bull knock out for galactose-alpha 1,3-galactose and N-glycolylneuraminic acid antigens

Author

Perota, Andrea, Lagutina, Irina, Duchi, Roberto, Zanfrini, Elisa, Lazzari, Giovanna, Judor, Jean Paul, Conchon, Sophie, Bach, Jean-Marie, Bottio, Tomaso, Gerosa, Gino, Costa, Cristina, Galiñanes, Manuel, Roussel, Jean Christian, Padler-Karavani, Vered, Cozzi, Emanuele, Soulillou, Jean Paul, Galli, Cesare, Avantea Laboratory of Reproductive Technologies, Fondazione Avantea [Cremona, Italy], Centre hospitalier universitaire de Nantes (CHU Nantes), Université de Nantes (UN), Immuno-Endocrinologie Cellulaire et Moléculaire, Institut National de la Recherche Agronomique (INRA)-Ecole Nationale Vétérinaire, Agroalimentaire et de l'alimentation Nantes-Atlantique (ONIRIS), Universita di Padova, IDIBELL--Hospital Universitari de Bellvitge, Vall d'Hebron University Hospital [Barcelona], Tel Aviv University [Tel Aviv], Transplant Immunology Unit, Universita degli Studi di Padova, European Project: 603049,EC:FP7:HEALTH,FP7-HEALTH-2013-INNOVATION-1,TRANSLINK(2013), ProdInra, Migration, Defining the role of xeno-directed and autoimmune events in patients receiving animal-derived bioprosthetic heart valves - TRANSLINK - - EC:FP7:HEALTH2013-09-01 - 2017-08-31 - 603049 - VALID, Institut National de la Recherche Agronomique (INRA)-École nationale vétérinaire, agroalimentaire et de l'alimentation Nantes-Atlantique (ONIRIS), Università degli Studi di Padova = University of Padua (Unipd), and Tel Aviv University (TAU)

Subjects
[SDV] Life Sciences [q-bio], [SDV]Life Sciences [q-bio], ComputingMilieux_MISCELLANEOUS
Abstract

International audience

Published
2019

8. Generation of cattle knockout for galactose‐α1,3‐galactose and N‐glycolylneuraminic acid antigens

Author

Perota, Andrea, Lagutina, Irina, Duchi, Roberto, Zanfrini, Elisa, Lazzari, Giovanna, Judor, Jean Paul, Conchon, Sophie, Bach, Jean Marie, Bottio, Tomaso, Gerosa, Gino, Costa Farré, Cristina, Galiñanes, Manuel, Roussel, Jean Christian, Padler-Karavani, Vered, Cozzi, Emanuele, Soulillou, Jean Paul, Galli, Cesare, Universitat Autònoma de Barcelona, Avantea Laboratory of Reproductive Technologies [Cremona, Italy], Fondazione Avantea [Cremona, Italy], Institut de transplantation urologie-néphrologie (ITUN), Université de Nantes (UN)-Centre hospitalier universitaire de Nantes (CHU Nantes), Immunoregulation And Immunointervention in Transplantation and Autoimmunity (Team 4 - U1064 Inserm - CRTI), Centre de Recherche en Transplantation et Immunologie (U1064 Inserm - CRTI), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN), Immuno-Endocrinologie Cellulaire et Moléculaire [Nantes] (IECM), Institut National de la Recherche Agronomique (INRA)-Université de Nantes (UN)-Ecole Nationale Vétérinaire, Agroalimentaire et de l'alimentation Nantes-Atlantique (ONIRIS), Department of Cardiac, Thoracic and Vascular Sciences and Public Health [Padova, Italy] (Cardiac Surgery Unit), Padova University School of Medicine [Padova, Italy] -Consorzio per la Ricerca Sanitaria - CORIS [Padova, Italy], Infectious Diseases and Transplantation Division [Barcelona, Spain], L’Hospitalet de Llobregat [Barcelona, Spain]-Bellvitge Biomedical Research Institute IDIBELL [Barcelona, Spain], Department of Cardiac Surgery & Reparative Therapy of the Heart [Barcelona, Spain], Vall d’Hebron Institute of Research [Barcelona, Spain]-Hospital Universitari Vall d'Hebron [Barcelona, Spain], Département de chirurgie thoracique et cardiovasculaire [CHU Nantes], Centre hospitalier universitaire de Nantes (CHU Nantes), Department of Cell Research and Immunology, Tel Aviv University [Tel Aviv], Transplantation Immunology Unit [Padua, Italy] (Department of Transfusion Medicine), University of Padua–Ospedale Giustinianeo [Padua, Italy], Le Bihan, Sylvie, Defining the role of xeno-directed and autoimmune events in patients receiving animal-derived bioprosthetic heart valves - TRANSLINK - - EC:FP7:HEALTH2013-09-01 - 2017-08-31 - 603049 - VALID, Immuno-Endocrinologie Cellulaire et Moléculaire (IECM), Institut National de la Recherche Agronomique (INRA)-Université de Nantes (UN)-Ecole Nationale Vétérinaire de Nantes-École nationale vétérinaire, agroalimentaire et de l'alimentation Nantes-Atlantique (ONIRIS), Università degli Studi di Padova = University of Padua (Unipd)-Consorzio per la Ricerca Sanitaria - CORIS [Padova, Italy], Vall d'Hebron University Hospital [Barcelona]-Vall d’Hebron Research Institute (VHIR), Tel Aviv University (TAU), Università degli Studi di Padova = University of Padua (Unipd), and European Project: 603049,EC:FP7:HEALTH,FP7-HEALTH-2013-INNOVATION-1,TRANSLINK(2013)

Subjects
Male, 0301 basic medicine, knockout, Neu5Gc, 030230 surgery, Epitope, Mixed Function Oxygenases, law.invention, Animals, Genetically Modified, Gene Knockout Techniques, chemistry.chemical_compound, 0302 clinical medicine, Carn de boví, N-Glycolylneuraminic acid, law, xenotransplantation, Polymerase chain reaction, 2. Zero hunger, Sanger sequencing, Heart valves, [SDV.MHEP] Life Sciences [q-bio]/Human health and pathology, Immunogenicity, Galactosyltransferases, Vàlvules cardíaques, Heart Valve Prosthesis, symbols, bioprosthetic Heart Valve (BHV), Somatic cell nuclear transfer, Xenotransplantation, Female, Original Article, Beef, cattle, CMAH, GGTA1, αGal, Food Hypersensitivity, Knockout, Transplantation, Heterologous, Immunology, Biology, 03 medical and health sciences, symbols.namesake, Antigens, Heterophile, Cytidine Monophosphate, Animals, Humans, Gene, Bioprosthesis, Galactosyltransferase, Transplantation, Galactose, Original Articles, Fibroblasts, Bioprosthetic Heart Valve (BHV), Molecular biology, 030104 developmental biology, chemistry, Cattle, Neuraminic Acids, [SDV.MHEP]Life Sciences [q-bio]/Human health and pathology
Abstract

International audience; Two well-characterized carbohydrate epitopes are absent in humans but present in other mammals. These are galactose-α1,3-galactose (αGal) and N-glycolylneuraminic acid (Neu5Gc) which are introduced by the activities of two enzymes including α(1,3) galactosyltransferase (encoded by the GGTA1 gene) and CMP-Neu5Gc hydroxylase (encoded by the CMAH gene) that are inactive in humans but present in cattle. Hence, bovine-derived products are antigenic in humans who receive bioprosthetic heart valves (BHVs) or those that suffer from red meat syndrome. Using programmable nucleases, we disrupted (knockout, KO) GGTA1 and CMAH genes encoding for the enzymes that catalyse the synthesis of αGal and Neu5Gc, respectively, in both male and female bovine fibroblasts. The KO in clonally selected fibroblasts was detected by polymerase chain reaction (PCR) and confirmed by Sanger sequencing. Selected fibroblasts colonies were used for somatic cell nuclear transfer (SCNT) to produce cloned embryos that were implanted in surrogate recipient heifers. Fifty-three embryos were implanted in 33 recipients heifers; 3 pregnancies were carried to term and delivered 3 live calves. Primary cell cultures were established from the 3 calves and following molecular analyses confirmed the genetic deletions. FACS analysis showed the double-KO phenotype for both antigens confirming the mutated genotypes. Availability of such cattle double-KO model lacking both αGal and Neu5Gc offers a unique opportunity to study the functionality of BHV manufactured with tissues of potentially lower immunogenicity, as well as a possible new clinical approaches to help patients with red meat allergy syndrome due to the presence of these xenoantigens in the diet.

Published
2019
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9. Introduction of the TLA sequencing analyses for a better identification of the transgene integrations, to select porcine founder animals for breeding

Author

Perota, Andrea, Mourad, Nizar, Lagutina, Irina, Duchi, Roberto, Zanfrini, Elisa, Lazzari, Giovanna, Gianello, Pierre, Galli, Cesare, 15th Congress of the International Xenotransplantation Association (IXA 2019), UCL - SSS/IREC/CHEX - Pôle de chirgurgie expérimentale et transplantation, and UCL - (SLuc) Service de chirurgie et transplantation abdominale

Abstract

Xenotransplantation of modified pig islets could be a possible medical treatment for T1/T2 diabetic patients. Tailored genetic engineering of a genomic safe harbor locus assisted by programmable nucleases (ZFNs, TALENs and CRISPR/Cas9) is making easier and reproducible also the generation of new transgenic large animal models. To validate in vivo our preliminary in vitro data (Mourad et al., 2017), we generated cloned transgenic porcine lines targeting the Insulin-GLP1Ser8M3R transgene (I-GS8M3R) into the genomic safe harbor V2G locus (Chromosome 9-EP2921048), using TALENs. Resulting transgenic pancreatic islets presented an increased glucose-stimulated insulin secretion. The aim of our work was to detect all the possible transgene integrations occurred in our targeted V2G/I-GS8M3R founder lines, introducing the Targeted Locus Amplification (TLA) technique as a new support to our genotyping procedure. TALENs-mediated integrations of transgene I-GS8M3R in V2G locus were achieved nucleofecting two different PERV-C negative and low PERV-A/B lines (female and male). Resulting Puromycin-resistant colonies were genotyped with 4 different PCR assays to select ones, having an intact transgene copy and a targeted V2G-allele, as nuclear donors for the Somatic Cell Nuclear Transfer (SCNT) experiments. At birth tissue biopsies were taken from alive piglets for genotyping analyses, identifying the donor colony, and to establish primary fibroblast cell lines for cryopreservation. TLA sequencing analyses of selected founders were outsourced to Cergentis (Netherlands). We selected by PCR, 6 V2G-targeted colonies (1 female = 1D5; 5 males = 1C2, 1F2, 2C2, 2E3 and 2E7) for SCNT. V2G-targeting of InsGLP1Ser8-M3R transgene resulted monoallelic in colonies 1D5, 2C2, 2E3 and 2E7 and bi-allelic in colonies 1C2 and 1F2. Characterization of alive born piglets revealed that colonies 1D5, 1C2, 1F2 and 2E3 produced healthy animals. TLA sequencing analyses confirmed the V2G-targeting of the I-GSer8M3R expression cassette for the male clones (1C2, 1F2 and 2E3), identifying also different transgenes tandems in lines 1C2 and 1F2. Instead, the TLA results on the 1D5 female line, revealed that an unexpected rearrangement occurred eliminating the I-GS8M3R transgene from the targeted V2G locus and that an intact copy of the I-GSer8M3R was randomly integrated only into the Chromosome 17. Conclusion: mono- or bi-allelic V2G-gene targeting of the I-GSer8M3R construct using TALENs was suitable for the generation of healthy pig clones. At the same time the access to the Next Generation Sequencing technologies benefits, integrating the TLA analyses in our classical molecular PCR screenings, permitted us to define better the characterization of the V2G-targeting events and to detect new random transgene integrations. Following this strategy, we will be able to select the founder animals suited for establishing a breeding colony.

Published
2019

11. Use of CRISPR/Cas9 technology combined with ssODNs to obtain a specific mono-allelic amino acid substitution in the TDP43 protein for the generation of a swine model of Amyotrophic Lateral Sclerosis (ALS)

Author

Quadalti, Corinne, Perota, Andrea, Lagutina, Irina, Lazzari, Giovanna, Crociara, Paola, Chieppa, Maria Novella, Cristina Casalone, Corona, Cristiano, Bonetto, Valentina, Galli, Cesare, Quadalti, C., Perota, A., Lagutina, I., Lazzari, G., Crociara, P., Chieppa, M. N., Casalone, C., Corona, C., Bonetto, V., and Galli, C

Subjects
CRISPR/Cas9, TDP43, pig model
Published
2016

13. Immune phenotype and IgG characteristics of Neu5Gc and alpha-1-3-gal double knock-out pigs

Author

Salama, Apolline, Conchon, Sophie, Perota, Andrea, Martinet, Bernard, Judor, Jean-Paul, Evanno, Gwénaëlle, Bernardet, Stéphanie Le-Bas, Le Berre, Ludmilla, Hervouet, Jeremy, Minault, David, Concordet, Jean-Paul, Dugast, Emilie, Vanhove, Bernard, Abadie, Jérôme, Gaide, Nicolas, Lagutina, Irina, Duchi, Roberto, Lazzari, Giovanna, Floch, Robert, Duvaux, Odile, Toutain, Pierre-Louis, Maillasson, Mike, Sachs, David H., Gauthier, Olivier, Brouard, Sophie, Cozzi, Emanuele, Blancho, Gilles, Perreault, Hélène, Bach, Jean-Marie, Galli, Cesare, Soulillou, Jean-Paul, Institut National de la Santé et de la Recherche Médicale (INSERM), Société d'Accélération du Transfert de Technologies (SATT OUEST VALORISATION), Fondazione Avantea, Partenaires INRAE, Xenothera [Nantes, France], Centre National de la Recherche Scientifique (CNRS), Animaux modèles pour la recherche en oncologie comparée (AMaROC), Ecole Nationale Vétérinaire, Agroalimentaire et de l'alimentation Nantes-Atlantique (ONIRIS), ToxAlim (ToxAlim), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Ecole Nationale Vétérinaire de Toulouse (ENVT), Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Ecole d'Ingénieurs de Purpan (INPT - EI Purpan), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Recherche Agronomique (INRA), Massachusetts General Hospital [Boston], University Hospital of Padua, Consortium for Research in Organ Transplantation (CORIT), University of Manitoba, University of Manitoba [Winnipeg], Immuno-Endocrinologie Cellulaire et Moléculaire (IECM), Ecole Nationale Vétérinaire de Nantes-Université de Nantes (UN)-Institut National de la Recherche Agronomique (INRA), Department of Veterinary Medical Sciences, University of Bologna, Institut National de la Recherche Agronomique (INRA)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Ecole Nationale Vétérinaire de Toulouse (ENVT), Université Fédérale Toulouse Midi-Pyrénées-Ecole d'Ingénieurs de Purpan (INPT - EI Purpan), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées, and ProdInra, Migration

Subjects
[SDV.OT]Life Sciences [q-bio]/Other [q-bio.OT], [SDV.OT] Life Sciences [q-bio]/Other [q-bio.OT], ComputingMilieux_MISCELLANEOUS
Abstract

International audience

Published
2015

14. Maternal age and in vitro culture affect mitochondrial number and function in equine oocytes and embryos

Author

Hendriks, W Karin, Colleoni, Silvia, Galli, Cesare, Paris, Damien B B P, Colenbrander, Ben, Roelen, Bernard A J, Stout, Tom A E, LS Klinische Reproductie, LS Voortplanting Inwendige Ziekten, Sub Celbiologisch lab., ES/FAH BRC, Fertility & Reproduction, LS Klinische Reproductie, LS Voortplanting Inwendige Ziekten, Sub Celbiologisch lab., ES/FAH BRC, Fertility & Reproduction, Hendriks, W.K., Colleoni, S., Galli, C., Paris, D.B.B.P., Colenbrander, B., Roelen, B.A.J., and Stout, T.A.E.

Subjects
Mitochondrial DNA, Embryonic Development, Reproductive technology, Biology, Mitochondrion, DNA, Mitochondrial, Oogenesis, Embryo Culture Techniques, Andrology, Endocrinology, Pregnancy, Genetics, Animals, Horses, Molecular Biology, equine oocyte, Maternal age, equine embryo, Embryo culture, Embryo, TFAM, mitochondrial, equines, In Vitro Oocyte Maturation Techniques, Mitochondria, in vitro colture, In vitro maturation, Reproductive Medicine, embryonic structures, Oocytes, Female, Animal Science and Zoology, Developmental Biology, Biotechnology
Abstract

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 (

Published
2015

16. Sus scrofa V2G: locus transgenico ad alta efficienza di integrazione e di espressione a lungo termine di transgeni nel suino

Author

Peverali AF, Biamonti G, Boncompagni E, Cubells M, Perota A, Lagutina I, LAZZARI, GIOVANNA, GALLI, CESARE, Peverali AF, Biamonti G, Boncompagni E, Cubells M, Lazzari G, Perota A, Lagutina I, and Galli C

Subjects
SUS SCROFA V2G, humanities, TRANSGENIC PIGS
Abstract

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.

Published
2014

17. Bovine Amniotic Fluid Mesenchymal Stem Cells characterization after culture in vitro

Author

ROSSI, BARBARA, MERLO, BARBARA, IACONO, ELEONORA, PAGLIARO, PASQUALE PAOLO, Tazzari, Pl, Ricci, F, GALLI, CESARE, Rossi, B, Merlo, B, Iacono, E, Pagliaro, Pp, Tazzari, Pl, Ricci, F, and Galli, C

Subjects
bovine, amniotic fluid, mesenchymal stem cells, characterization
Abstract

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.

Published
2014

18. Cloning of Equines

Author

GALLI, CESARE, LAZZARI, GIOVANNA, Lagutina I, Duchi R, Colleoni S, Cibelli J, Gurdon J, Wilmut I, Lanza M, West MD, Campbell KS, Galli C, Lagutina I, Duchi R, Colleoni S, and Lazzari G

Subjects
EQUINE, CLONING
Abstract

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).

Published
2014

19. Targeted RMCE-live piglets generated by SCNT following sequential double site-specific gene modifications of a porcine EGFP line

Author

Perota, Andrea, Lagutina, Irina, Duchi, Roberto, Quadalti, Corinne, Turini, Paola, Crotti, Gabriella, Colleoni, Silvia, Lazzari, Giovanna, Peverali, Fiorenzo, Franco Lucchini, Galli, Cesare, Perota, A., Lagutina, I., Duchi, R., Quadalti, C., Turini, P., Crotti, G., Colleoni, S., Lazzari, G., Peverali, F., Lucchini, F., and Galli, C

Subjects
RMCE - Recombinase mediated cassette exchange, Settore AGR/17 - ZOOTECNICA GENERALE E MIGLIORAMENTO GENETICO, recombination-mediated cassette exchange, porcine EGFP line, Transgenic swine
Published
2014

20. The Effect of Cilostamide on Gap Junction Communication Dynamics, Chromatin Remodeling, and Competence Acquisition in Pig Oocytes Following Parthenogenetic Activation and Nuclear Transfer

Author

Dieci C, Lodde V, Franciosi F, Lagutina I, Tessaro I, Modina SC, Albertini DF, Luciano AM, LAZZARI, GIOVANNA, GALLI, CESARE, Dieci C, Lodde V, Franciosi F, Lagutina I, Tessaro I, Modina SC, Albertini DF, Lazzari G, Galli C, and Luciano AM

Subjects
Cilostamide, Parthenogenetic Activation, Chromatin Remodeling, Pig oocyte, NUCLEAR TRANSFER, Gap Junction Communication Dynamic
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), 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.

Published
2013

21. Isolation and characterization of mesenchymal stem cells from bovine amniotic fluid at different gestational ages

Author

ROSSI, BARBARA, MERLO, BARBARA, IACONO, ELEONORA, GALLI, CESARE, Tazzari PL, Ricci F, Rossi B, Merlo B, Iacono E, Tazzari PL, Ricci F, and Galli C.

Subjects
bovine, amniotic fluid, mesenchymal stem cell
Abstract

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.

Published
2013

25. Stemness features in mesenchymal stem cells from equine amniotic fluid, Wharton’s jelly and umbilical cord blood

Author

ROSSI, BARBARA, MERLO, BARBARA, IACONO, ELEONORA, MARIELLA, JOLE, CASTAGNETTI, CAROLINA, GALLI, CESARE, S. Colleoni, B. Rossi, B. Merlo, E. Iacono, S. Colleoni, J. Mariella, C. Castagnetti, and C. Galli

Subjects
Umbilical cord blood, EQUINE, amniotic fluid, MESENCHYMAL STEM CELLS, WHARTON'S JELLY
Abstract

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.

Published
2013

28. Equine Assisted Reproduction and Embryo Technologies

Author

GALLI, CESARE, LAZZARI, GIOVANNA, Colleoni S, Duchi R, Lagutina I, Galli C, Colleoni S, Duchi R, Lagutina I, and Lazzari G

Subjects
REPRODUCTION, animal structures, EQUINE, embryonic structures, EMBRYO
Abstract

Equine Assisted Reproduction and Embryo Technologies

Published
2013

29. Fertilization and development of equine and swine oocytes following ICSI with refrigerated and frozen semen of fertile and infertile stallions

Author

Colleoni S, Duchi R, Baca Castex C, Lagutina I, LAZZARI, GIOVANNA, MARI, GAETANO, GALLI, CESARE, COLLEONI S, LAZZARI G, DUCHI R, BACA CASTEX C, MARI G, LAGUTINA I, GALLI C, Colleoni S, Lazzari G, Duchi R, Baca Castex C, Mari G, Lagutina I, and Galli C

Subjects
EQUINE, OOCYTES, Fertilization, STALLION, SWINE, INTRACYTOPLASMIC SPERM INJECTION, ICSI
Published
2012

30. Novel transgenic animal model of Amyotrophic Lateral Sclerosis

Author

Cristina CASALONE, Maria Novella CHIEPPA, Cristiano CORONA, Caterina BENDOTTI, Andrea PEROTA, Maria CARAMELLI, Irina LAGUTINA, GALLI, CESARE, LAZZARI, GIOVANNA, Cristina CASALONE, Cesare GALLI, Maria Novella CHIEPPA, Cristiano CORONA, Caterina BENDOTTI, Andrea PEROTA, Giovanna LAZZARI, Maria CARAMELLI, and Irina LAGUTINA

Subjects
SUINO, SLA, humanities
Abstract

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

Published
2012

39. Pig to primate lamellar corneal xenograft : first use of Gal-KO and transgenic pig donors

Author

B. Vabres, S. Le Bas Bernardet, A. Pallier, S. Brouard, D. Minault, J. Hervouet, Y. Ducourneau, A. Moreau, E. Cozzi, J. P. Soulillou, G. Blancho, GALLI, CESARE, B. Vabre, S. Le Bas-Bernardet, A. Pallier, S. Brouard, D. Minault, J. Hervouet, Y. Ducourneau, A. Moreau, C. Galli, E. Cozzi, J.P. Soulillou, and G. Blancho

Subjects
Gal-KO pig, corneal xenograft, TRANSGENIC PIGS
Published
2011

40. Porcine galactosyl-transferase knock-out transgenic for human CD55, CD59, CD39 kidneys are actually rejected by baboons despite plasma/B cells and complement blockade

Author

S. Le Bas Bernardet, J. Branchereau, X. Tillou, J. Hervouet, D. Minault, N. Dilek, M. Chatelais, B. Charreau, K. Renaudin, P. Cowan, A. d’Apice, E. Cozzi, J. P. Soulillou, G. Blancho, GALLI, CESARE, S. Le Bas-Bernardet, J. Branchereau, X. Tillou, J. Hervouet, D. Minault, N. Dilek, M. Chatelai, B. Charreau, K. Renaudin, P. Cowan, A. d’Apice, C. Galli, E. Cozzi, J.P. Soulillou, and G. Blancho

Subjects
XENOTRANSPLANTATION, TRANSGENIC PIGS, BABOON
Published
2011

41. Preformed non-Gal antibodies are highly cytotoxic even against Galactosyl-transferase knockout, human CD55, CD59, CD39 transgenic pig organs in a model of kidney transplantation into baboons

Author

S. Le Bas Bernardet, N. Poirier, X. Tillou, J. Hervouet, D. Minault, K. Renaudin, N. Dilek, M. Chatelais, B. Charreau, J. Branchereau, P. Cowan, A. d’Apice, E. Cozzi, J. P. Soulillou, G. Blancho, GALLI, CESARE, S. Le Bas-Bernardet, N. Poirier, X. Tillou, J. Hervouet, D. Minault, K. Renaudin, N. Dilek, M. Chatelai, B. Charreau, J. Branchereau, P. Cowan, A. d’Apice, C. Galli, E. Cozzi, J.P. Soulillou, and G. Blancho

Subjects
Preformed non-Gal antibodie, TRANSGENIC PIGS, BABOON
Published
2011

42. Improved cloning efficiency using hCD55/alpha 1,3-Gal knock-out cell clones selected for high hCD55 expression level

Author

Perota, Andrea, Lagutina, Irina, Colleoni, Silvia, Duchi, Roberto, Lazzari, Giovanna, Cozzi, Emanuele, Calabrese, Fiorella, Chatelais, Mathias, Charreau, Beatrice, Franco Lucchini, Galli, Cesare, A. Perota, I. Lagutina, S. Colleoni, R. Duchi, G. Lazzari, E. Cozzi, F. Calabrese, M. Chatelai, B. Charreau, F. Lucchini, and C. Galli

Subjects
transgenic pig, Somatic cell nuclear transfer, hCD55/alpha 1, PIG, Settore VET/02 - FISIOLOGIA VETERINARIA, xenotransplantation, 3-Gal knock-out cell
Published
2011

46. X trisomy in a sterile mare

Author

de Lorenzi L, Molteni L, Zannotti M, Parma P., GALLI, CESARE, de Lorenzi L, Molteni L, Zannotti M, Galli C, and Parma P

Subjects
endocrine system, EQUINE, X Chromosome, Karyotyping, X-TRISOMY, Animals, Female, Trisomy, Horses, Infertility, Female, In Situ Hybridization, Fluorescence, INFERTILITY
Abstract

This report concerns the cytogenetic analysis, using both C-banding and fluorescence in situ hybridisation techniques, of a sterile mare. Results obtained revealed a 2n = 65, XXX condition with no sign of mosaicism. The work supports the suggestion that X trisomy, rare in horse, causes infertility in mares and is not associated to other clearly visible phenotypic features.

Published
2010

49. Production and characterization of Gal-/- minipigs over-expressing hCD55

Author

Brunetti, Dario, Perota, Andrea, Lagutina, Irina, Chatelais, Mathias, Charreau, Beatrice, Duchi, Roberto, Lazzari, Giovanna, Franco Lucchini, and Galli, Cesare

Subjects
transgenic pig, Gal knockout, Settore VET/03 - PATOLOGIA GENERALE E ANATOMIA PATOLOGICA VETERINARIA
Published
2010

50. Production and characterization of GAL-/- minipigs over-expressing hCD55

Author

Brunetti D., Perota A., Lagutina I. Chatelais M., Charreau B., Duchi R., Lucchini F., LAZZARI, GIOVANNA, GALLI, CESARE, Brunetti D., Perota A., Lagutina I. Chatelais M., Charreau B., Duchi R., Lazzari G., Lucchini F., and Galli C.

Subjects
GAL-/- minipigs, hCD55
Published
2010

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