Systematic discovery of transcription factors that improve hPSC-derived cardiomyocyte maturation via temporal analysis of bioengineered cardiac tissues.

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have the potential to become powerful tools for disease modeling, drug testing, and transplantation; however, their immaturity limits their applications. Transcription factor (TF) overexpression can improve hPSC-CM maturity, but identifying these TFs has been elusive. Toward this, we establish here an experimental framework for systematic identification of maturation enhancing factors. Specifically, we performed temporal transcriptome RNAseq analyses of progressively matured hPSC-derived cardiomyocytes across 2D and 3D differentiation systems and further compared these bioengineered tissues to native fetal and adult-derived tissues. These analyses revealed 22 TFs whose expression did not increase in 2D differentiation systems but progressively increased in 3D culture systems and adult mature cell types. Individually overexpressing each of these TFs in immature hPSC-CMs identified five TFs (KLF15, ZBTB20, ESRRA, HOPX, and CAMTA2) as regulators of calcium handling, metabolic function, and hypertrophy. Notably, the combinatorial overexpression of KLF15, ESRRA, and HOPX improved all three maturation parameters simultaneously. Taken together, we introduce a new TF cocktail that can be used in solo or in conjunction with other strategies to improve hPSC-CM maturation and anticipate that our generalizable methodology can also be implemented to identify maturation-associated TFs for other stem cell progenies.