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The therapeutic potential of embryonic stem cells (ESCs) in ischaemic heart disease has been widely explored. However, tumourigenesis upon implantation interferes with the clinical application of ESC transplantation. This study aims to evaluate the influence of differentiation and enrichment of transplanted ESCs on tumourigenesis in infarcted rat hearts.Mouse ESCs (mESCs) were cultured using a bioreactor system to develop embryoid bodies, which were then induced with 1% ascorbic acid to differentiate into cardiomyocytes. The mESCs-derived cardiomyocytes (mESCs-CMs) were enriched by Percoll density gradient separation. The specific markers (OCT-4, Sox2, and Nanog) of undifferentiated ESCs were detected by PCR both in mESCs and in mESCs-CMs, but not in the mESC-derived Percoll-enriched cardiomyocytes (mESC-PE-CMs). Immunosuppressed rats with infarcted hearts were randomly injected with the mESCs, mESC-CMs, or mESC-PE-CMs. Eight weeks after cell transplantation, histological and immunohistochemical analysis showed that the transplantation of both mESCs and mESC-CMs caused the formation of teratomas. The incidence of teratoma was markedly lower (P < 0.05) in the mESC-CMs group than in the mESCs group. The average tumour volume was significantly lower (P < 0.05) in the mESC-CMs group than in the mESCs group. Tumour formation was absent in the mESC-PE-CMs group.Enrichment of the mESC-differentiated cardiomyocytes inhibited the development of teratoma after cell transplantation in the infarcted rat hearts. These findings offer a new strategy for eliminating teratoma formation in ESCs transplantation and could be a step forward in the development of human ESCs transplantation therapy in ischaemic heart disease.