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Background  Cardiomyocytes differentiated from human pluripotent stem cells can serve as an unexhausted source for a cellular cardiac disease model. Although small molecule–mediated cardiomyocyte differentiation methods have been established, the differentiation efficiency is relatively unsatisfactory in multiple lines due to line‐to‐line variation. Additionally, hurdles including line‐specific low expression of endogenous growth factors and the high apoptotic tendency of human pluripotent stem cells also need to be overcome to establish robust and efficient cardiomyocyte differentiation.    Methods and Results  We used the H9–human cardiac troponin T–e GFP  reporter cell line to screen for small molecules that promote cardiac differentiation in a monolayer‐based and growth factor–free differentiation model. We found that collaterally treating human pluripotent stem cells with rapamycin and  CHIR 99021 during the initial stage was essential for efficient and reliable cardiomyocyte differentiation. Moreover, this method maintained consistency in efficiency across different human embryonic stem cell and human induced pluripotent stem cell lines without specifically optimizing multiple parameters (the efficiency in H7, H9, and  UQ 1 human induced pluripotent stem cells is 98.3%, 93.3%, and 90.6%, respectively). This combination also increased the yield of cardiomyocytes (1:24) and at the same time reduced medium consumption by about 50% when compared with the previous protocols. Further analysis indicated that inhibition of the mammalian target of rapamycin allows efficient cardiomyocyte differentiation through overcoming p53‐dependent apoptosis of human pluripotent stem cells during high‐density monolayer culture via blunting p53 translation and mitochondrial reactive oxygen species production.    Conclusions  We have demonstrated that mammalian target of rapamycin exerts a stage‐specific and multifaceted regulation over cardiac differentiation and provides an optimized approach for generating large numbers of functional cardiomyocytes for disease modeling and in vitro drug screening.

Rapamycin and CHIR 99021 Coordinate Robust Cardiomyocyte Differentiation From Human Pluripotent Stem Cells Via Reducing p53‐Dependent Apoptosis