2013 - CTS 2013 Congress

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Oral Communications 9

20.3 - Mesenchymal stromal cells isolated from green fluorescent protein transgenic pigs: a new large animal model for translational studies

Presenter: Tommaso, Dominioni, Pavia, Italy
Authors: Lorenzo Cobianchi1,2,3,4, Jacopo Viganò1,2,3,4, Maria Antonietta Avanzini1,2,3,4, Melissa Mantelli1,2,3,4, Daniela Ingo1,2,3,4, Tommaso Dominioni1,2,3,4, Maria Ester Bernardo1,2,3,4, Michela Demartino1,2,3,4, Alberto Fratti1,2,3,4, Sara Baleri1,2,3,4, Riccardo Rizzo1,2,3,4, Alessandro Spolini1,2,3,4, Francesco Calabrese1,2,3,4, Edoardo Segalini1,2,3,4, Giorgio Querini1,2,3,4, Andrea Bottazzi1,2,3,4, Paolo Dionigi1,2,3,4, Mario Alessiani1,2,3,4, Sandro Zonta1,2,3,4

Mesenchymal stromal cells isolated from green fluorescent protein transgenic pigs: a new large animal model for translational studies

Lorenzo Cobianchi1,2,3,4, Jacopo Viganò1,2,3,4, Maria Antonietta Avanzini1,2,3,4, Melissa Mantelli1,2,3,4, Daniela Ingo1,2,3,4, Tommaso Dominioni1,2,3,4, Maria Ester Bernardo1,2,3,4, Michela Demartino1,2,3,4, Alberto Fratti1,2,3,4, Sara Baleri1,2,3,4, Riccardo Rizzo1,2,3,4, Alessandro Spolini1,2,3,4, Francesco Calabrese1,2,3,4, Edoardo Segalini1,2,3,4, Giorgio Querini1,2,3,4, Andrea Bottazzi1,2,3,4, Paolo Dionigi1,2,3,4, Mario Alessiani1,2,3,4, Sandro Zonta1,2,3,4

1Surgery, University of Pavia, IRCCS Policlinico San Matteo, Pavia, Italy; 2Oncoematologia Pediatrica, IRCCS Fondazione Policlinico San Matteo, Pavia, Italy; 3Dipartimento di Ematologia ed Oncologia, IRCCS Ospedale Bambino Gesù, Roma, Italy; 4Rianimazione 2, IRCCS Fondazione Policlinico San Matteo, Pavia, Italy

 

Large animals, pig in particular, have emerged as an important tool for development of surgical techniques, advancement of xenotransplantation, creation of important disease models, and preclinical testing of novel cell therapies. Large animal models play a crucial role to investigate the biological and functional properties of Mesenchymal Stem Cells (MSCs) as novel cellular therapy.
Transgenic swine has been generated through genetic engineering and somatic cell nuclear transfer that will express EGFP (Enanched Green Fluorescente Protein) at high level in somatic cells in order to have a specific source of marked MSCs.
Porcine MSCs (pMSCs) were isolated from 6 EGFP-transgenic pigs, expanded ex vivo, characterized by flow cytometry and assessed for their differentiation capacities. EGFP-enhanched pMSCs resulted positive for CD90, CD29 and CD105. The proliferative capacity was evaluated in terms of population doubling from passage P1 to P4 and compared to hMSCs. We also evaluated the proliferation of porcine and human PBMCs stimulated with phytohemagglutinin in the presence of autologus and allogeneic pMSCs or hMSCs [1-3].
EGFP-enhanced pMSCs were traced through the identification of the EGFP by direct visualisation under blue light or by immunohystochemistry. We evaluated also the level of interleukin-6 in colture supernatants collected at the end of PHA proliferation assay in the autologus and allogeneic porcine settings, both porcine settings showed a strong increase in pIL-6 levels after EGFP-enhanched pMSC addition whereas IL-10 and TGF-beta  were undetectable[4].
We have demonstrated that the methods for harvesting, culturing and ex vivo expanding hMSCs can be successfully reproduced in EGFP transgenic pigs. EGFP-enhanched pMSCs and hMSCs have similarities in terms of morphology, immunophenotype and differentiation potential. We also documented that EGFP-enhanched pMSCs have the capacity to inhibit the proliferation of pPBMCs in both the autologus and allogeneic settings. Our study provides evidence that green fluorescent pigs represent a very interesting animal model with a lot of potential applications in the field of cellular transplantation. 
1. Avanzini, M.A.et Al. Generation of mesenchymal stromal cells in the presence of platelet lysate: a phenotypical and functional comparison of umbilical cord blood-            and            bone            marrow-derived progenitors. Haematologica 2009; 94: 1649-1660.
 
2. Bernardo, M.E., et Al. Phenotypical/functional characterization of in vitro expanded mesenchymal stromal cells from Crohn’s disease patients. Cytotherapy 2009; 11: 825-836
 
3. Ball, L.M, et Al. Co- transplantation of ex-vivo expanded mesenchymal stem cells accelerates lymphocyte recovery and may reduce the risk of graft failure in haploidentical hematopoietic stem cell transplantation. Blood 2007; 110: 2764-2767
 
4. Comite, P. et Al. Isolation and ex vivo expansion of bone marrow-derived porcine mesenchymal stromal cells: potential for application in an experimental model of solid organ transplantation in large animals. Transplant. Proc. 2010; 42: 1341-1343

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