A Direct Comparison Of Cardiomyocyte Maturation In Vivo And In Vitro At Single Cell Resolution

Pluripotent stem cell-derived cardiomyocytes (PSC-CMs) offer vast potential for clinical applications including cell therapy, drug screening, and disease modeling. However, in vitro -derived PSC-CMs incompletely mature with fetal-like phenotype, which drastically limits their clinical applications. Currently, it remains unknown why PSC-CMs fail to fully mature in culture. We sought to understand CM maturation process by generating high-quality single-cell RNA-sequencing (scRNA-seq) reference of in vivo mouse CMs sampled from embryonic stage to adult stage. Subsequently, we generated isogenic embryonic stem cells and established an in vitro scRNA-seq reference of differentiated CMs across a range of differentiation timepoints. By directly comparing the in vivo and the in vitro maturation trajectories, we identified a group of dysregulated transcription factors (TFs), NFE2L2, NRF1, MEF2A, JUN, YY1, ESRRA, SRF, SOX9, PPARA, which may be important to activate the CM maturation program. Therefore, we increased their levels in PSC-CMs and subsequently examined their transcriptome changes. Our initial analysis showed that higher levels of the TFs increase the expression of electron transport chain protein genes and decrease the expression of glycolysis protein genes, suggesting the initiation of metabolic maturation. Our study presents a first direct comparison of CM maturation in vivo and in vitro at single-cell resolution, which illuminates new research directions for understanding PSC-CM maturation.