Differentiation “In Vitro” of Primary and Immortalized Porcine Mesenchymal Stem Cells Into Cardiomyocytes for Cell Transplantation I. Moscoso, A. Centeno, E. López, J.I. Rodriguez-Barbosa, I. Santamarina, P. Filgueira, M.J. Sánchez, R. Domı´nguez-Perles, G. Peñuelas-Rivas, and N. Domenech ABSTRACT Cell transplantation to regenerate injured tissues is a promising new treatment for patients suffering several diseases. Bone marrow contains a population of progenitor cells known as mesenchymal stem cells (MSCs), which have the capability to colonize different tissues, replicate, and differentiate into multilineage cells. Our goal was the isolation, character- ization, and immortalization of porcine MSCs (pMSCs) to study their potential differen- tiation “in vitro” into cardiomyocytes. pMSCs were obtained from the aspirated bone marrow of Large-White pigs. After 4 weeks in culture, adherent cells were phenotypically characterized by flow cytometry and immunochemistry by using monoclonal antibodies. Primary pMSCs were transfected with the plasmid pRNS-1 to obtain continuous growing cloned cell lines. Fresh pMSCs and immortalized cells were treated with 5-azacytidine to differentiate them into cardiomyocytes. Flow cytometry analysis of isolated pMSCs demonstrated the following phenotype, CD90 pos , CD29 pos , CD44 pos , SLA-I pos , CD106 pos , CD46 pos and CD45 neg , CD14 neg , CD31 neg , and CD11b neg , similar to that described for human MSC. We derived several stable immortalized MSC cell lines. One of these, called pBMC-2, was chosen for further characterization. After “in vitro” stimulation of both primary or immortalized cells with 5-azacytidine, we obtained different percentages (30%–50%) of cells with cardiomyocyte characteristics, namely, positive for -Actin and T-Troponin. Thus, primary or immortalized pMSCs derived from bone marrow and cultured were able to differentiate “ex vivo” into cardiac-like muscle cells. These elements may be potentials tools to improve cardiac function in a swine myocardial infarct model. M YOCARDIAL infarction is one of the primary cause of death in developed countries. Cellular therapy to regenerate cardiomyocytes is a new therapy. 1 An important consideration to the transplanted choice of cell. Myocardial transplants of cultured heart cells, skeletal myoblast, 2 or smooth muscle cells have been shown to prevent heart failure after myocardial injury. One particularly promising cell alternative for transplantation is mesenchymal stem cells (MSCs) derived from bone marrow. Human MSCs have been isolated and cultivated, thereafter differentiating into cells with similar characteristics as heart myocytes. 3 Those cells show a stable phenotype that grows as a monolayer “in vitro.” In the same way MSCs of various species have been shown to undergo myogenic differentia- tion. 4,5 To provide clinical insights for cell therapy, key experiments must be carried out in large animals. Recently, there is growing interest in the pig as an animal model for biomedical studies. 6 In this study, we show that porcine MSCs (pMSCs) can be isolated from porcine bone marrow, readily expanded in culture, immortalized, and induced with 5-azatidine to differentiate “in vitro” into myogenic cells, which express cardiac muscle cell markers. Thus, From the Unidad de Investigacion, Complejo Hospitalario Universitario, Juan Canalejo, A Coruña, and Unidad de Investi- gación en Transplante, Hospitalario Universitario, Virgen de la Arrixaca, Murcia, Spain. Supported by Xunta de Galicia, Santiago de Compostela (Expediente PGIDT03SAN91604PR1), Spain. Address reprint requests to Nieves Domenech, PhD, Unidad de Investigacion, Complejo Hospitalario Universitario Juan Ca- nalejo, As Xubias s/n. 15006, A Coruña, Spain. E-mail: ndomgar@canalejo.org © 2005 by Elsevier Inc. All rights reserved. 0041-1345/05/$–see front matter 360 Park Avenue South, New York, NY 10010-1710 doi:10.1016/j.transproceed.2004.12.247 Transplantation Proceedings, 37, 481– 482 (2005) 481