Transcriptome and epigenome dynamics of the clonal heterogeneity of human induced pluripotent stem cells for cardiac differentiation

Human induced pluripotent stem cells (hiPSCs) generate multiple clones with inherent heterogeneity, leading to variations in their differentiation capacity. Previous studies have primarily addressed line-to-line variations in differentiation capacity, leaving a gap in the comprehensive understanding of clonal heterogeneity. Here, we aimed to profile the heterogeneity of hiPSC clones and identify predictive biomarkers for cardiomyocyte (CM) differentiation capacity by integrating transcriptomic, epigenomic, endogenous retroelement, and protein kinase phosphorylation profiles. We generated multiple clones from a single donor and validated that these clones exhibited comparable levels of pluripotency markers. The clones were classified into two groups based on their differentiation efficiency to CMs-productive clone (PC) and non-productive clone (NPC). We performed RNA sequencing (RNA-seq) and assay for transposase-accessible chromatin with sequencing (ATAC-seq). NPC was enriched in vasculogenesis and cell adhesion, accompanied by elevated levels of phosphorylated ERK1/2. Conversely, PC exhibited enrichment in embryonic organ development and transcription factor activation, accompanied by increased chromatin accessibility near transcription start site (TSS) regions. Integrative analysis of RNA-seq and ATAC-seq revealed 14 candidate genes correlated with cardiac differentiation potential. Notably, TEK and SDR42E1 were upregulated in NPC. Our integrative profiles enhance the understanding of clonal heterogeneity and highlight two novel biomarkers associated with CM differentiation. This insight may facilitate the identification of suboptimal hiPSC clones, thereby mitigating adverse outcomes in clinical applications.