摘要The recent breakthrough in the generation of rat embryonic stem cells (rESCs) opens the door to application of gene targeting to create models for the study of human diseases.In addition,the in vitro differentiation system from rESCs into derivatives of three germ layers will serve as a powerful tool and resource for the investigation of mammalian development,cell function,tissue repair,and drug discovery.However,these uses have been limited by the difficulty of in vitro differentiation.The aims of this study were to establish an in vitro differentiation system from rESCs and to investigate whether rESCs are capable of forming terminal-differentiated cardiomyocytes.Using newly established rESCs,we found that embryoid body (EB)-based method used in mouse ESC (mESC) differentiation failed to work for the serum-free cultivated rESCs.We then developed a protocol by combination of three chemical inhibitors and feeder-conditioned medium.Under this condition,rESCs formed EBs,propagated and differentiated into three embryonic germ layers.Moreover,rESC-formed EBs could differentiate into spontaneously beating cardiomyocytes after plating.Analyses of molecular,structural,and functional properties revealed that rESC-derived cardiomyocytes were similar to those derived from fetal rat hearts and mESCs.In conclusion,we successfully developed an in vitro differentiation system for rESCs through which functional myocytes were generated and displayed phenotypes of rat fetal cardiomyocytes.This unique cellular system will provide a new approach to study the early development and cardiac function,and serve as an important tool in pharmacological testing and cell therapy.