Transcriptome and epigenome dynamic of the clonal heterogeneity of human induced pluripotent stem cells for cardiac differentiation [ATAC-Seq]

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 identify predictive biomarkers for cardiomyocyte 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 pluripotent markers. These clones were then classified into two groups based on their differentiation capacity: productive clone (PC) and non-productive clone (NPC). We performed RNA sequencing and assay for transposase-accessible chromatin with sequencing (ATAC-seq). NPC was enriched in vasculogenesis and cell adhesion, accompanied by elevated ERK1/2 signaling pathway phosphorylation. Conversely, PC exhibited enrichment in embryonic organ development and transcription factor activation, accompanied by increased chromatin accessibility. Integrative analysis of RNA sequencing and ATAC-seq revealed 14 candidate genes correlated with cardiac differentiation propensity. Notably, TEK and SDR42E1 were upregulated in NPC. Our integrative profiles enhance the understanding of clonal heterogeneity and highlight two novel biomarkers associated with cardiomyocytes differentiation. This insight may facilitate the identification of suboptimal hiPSC clones, thereby mitigating adverse outcomes in clinical applications. Overall design: To assess the differentiation potential of hiPSC clones, we quantified TNNT2-positive cells using flow cytometry between days 12 to 15 of cardiomyocytes (CMs) differentiation. Notably, we observed diverse proportions among the clones, with some consistently exhibiting robust differentiation rates (>60%), while others displayed lower rates (<50%) along with a higher proportion of non-beating cells. This distinct pattern facilitated the categorization of certain clones as productive and others as non-productive based on their respective differentiation rates, indicative of varying capacities for CMs differentiation.These findings highlighted the presence of a heterogeneous population encompassing both CMs and non-CMs among the hiPSC clones, suggesting differing potentials for differentiation. Therefore, we plan to explore the underlying variability by examining genetic and epigenetic influences on differentiation potential using techniques such as RNA-seq and ATAC-seq. Additionally, to characterize the gene expression and chromatin accessibility landscapes comprehensively across all studied hiPSC clones, we will conduct integrative RNA sequencing (RNA-seq) and assay for transposase-accessible chromatin by sequencing (ATAC-seq) analyses.