Cardiomyocytes expressing host markers, such as the Y chromosome in sex-mismatched transplants, have been described in human allografts, suggesting that circulating cells can contribute to cardiac regeneration. It has not been established, however, whether host-derived cardiomyocytes result from transdifferentiation of stem cells or cell fusion. To address this issue, we used heterotopic heart xenografts and looked for markers of donor and recipient cells. Golden Syrian hamsters or transgenic mice expressing nuclear beta-galactosidase under the control of the cardiac troponin I promoter served as organ donors, while GFP⁺ transgenic rats were used as recipients. GFP⁺ cells, including abundant CD-45⁺ inflammatory cells and rare undifferentiated cells expressing early cardiac markers (GATA-4 or MEF2C), were found in xenografts harvested two weeks after surgery. In addition, rare GFP⁺ mature cardiomyocytes were found in 7 of 8 hamster xenografts and 6 of 6 mouse xenografts. The proportion of these cells was very low (0.0001% to 0.0344% in hamster xenografts) but similar to the one observed in control rat heart allografts. Without exception, all GFP⁺ cardiomyocytes also expressed donor markers, i.e., hamster membrane antigens or lacZ, so they must derive from cell fusion, not transdifferentiation.-- Dedja, A., Zaglia T., Dall’Olmo, L., Chioato, T., Thiene, G., Fabris, L., Ancona, E., Schiaffino, S., Ausoni, S., Cozzi, E. Hybrid cardiomyocytes derived by cell fusion in heterotopic cardiac xenografts.