Congenital heart defects (CHDs) affect 1% of live new-borns and corrective surgery is usually the only therapy available. Truncus Arteriosus (TA) is CHD characterized by a common aorta/pulmonary artery and ventricular septal defect. Right outflow tract reconstruction in TA patients involves the use of cardiac patches to attach replacement grafts to the myocardium. Current patch materials are prone to calcification, immunogenicity and lack of growth. The aim of this study is to engineer a cardiac patch using patient’s stem cells and a suitable scaffold. Mesenchymal Stem Cells (MSC) were isolated, by enzymatic digestion, from neonatal (P1) piglet thymus (T) and expanded in vitro. Histological staining, viability/cytotoxicity assay and scanning electron microscopy (SEM) have been performed to establish cells’ ability to populate and eventually differentiate onto the scaffold. We have first assessed the potential of a clinically used scaffold, made of an acellular matrix, to support cell growth and proliferation once seeded. Cardiomyocytes, isolated by enzymatic digestion from neonatal rat (P2) hearts, were seeded on the scaffold and used as a control. Both TMSCs and rat cardiomyocytes seeded on the acellular matrix have engrafted and proliferated on it. Only few dead cells were detected. A confluent and oriented monolayer of cells was observed by SEM, suggesting that the acellular matrix represents a good scaffold for the growth of the cells of interest. As there is evidence that MSCs can differentiate into cardiomyocyte-like cells, we attempted to commit TMSCs into cardiac muscle phenotype. We have first performed the differentiation in a standard bi-dimensional culture system. TMSCs have been submitted to distinct differentiation-inducing media: a conditioned medium obtained by separate cultures of neonatal rat cardiomyocytes and normal media supplemented with different factors, such as 5-Azacytidine, Transforming Growth Factor-β1, Ascorbic Acid. Expression of Myosin Heavy Chain (MHC), Sarcomeric α-Actinin and Desmin was investigated by immunocytochemistry and quantified with Western Blot. Expression of the cardiac-specific genes GATA-4, Nkx 2.5, α-Actin and Desmin was analysed by RT- PCR. Results show that after 3 weeks of differentiation TMSCs express some cardiac markers. Positivity for MHC, Sarcomeric α-Actinin and Desmin was detected. The mRNA of GATA-4, α-Actin and Desmin showed expression increase in myogenic cells compared with untreated TMSCs. Our data confirm the ability of TMSCs to differentiate into cardiomyocyte-like cells in a bi-dimensional system. As we demonstrate that TMSCs can populate the acellular scaffold, it is likely that a differentiation process could also be triggered on it. These observations imply that TMSCs can be considered as a source of cells clinically relevant to engineer cardiac patches.