Abstract 145: Isolation & Characterization of Heart-field Specific Cardiomyocytes

Abstract

Cardiovascular disease (CVD) is the leading cause of death worldwide. Human embryonic stem cell (hESC)-derived cardiovascular progenitors (CVPs) or cardiomyocytes (CMs) represent a promising candidate for cell-based therapies to treat CVD. Myocardial infarction leads to extensive CM death mainly within the left ventricle, which is predominantly derived from the first heart field (FHF) during embryonic development. We postulate that the generation of chamber-specific CMs may play a key role in the development of safe and efficacious regenerative therapy. As a first step, we generated and characterized a FHF-specific TBX5-TdTomato +/W hESC reporter line. We show that TBX5 + cells represent an enriched population of FHF CVPs that can give rise to CMs, endothelial, and smooth muscle cells in vitro . Interestingly, we observed that TBX5 - cells can also generate contractile CMs. Bulk RNA-sequencing analysis at different stages of development suggested that TBX5 - cells are enriched for second heart field (SHF) CMs. To enable prospective isolation of FHF and SHF CMs, we generated a double transgenic TBX5-TdTomato +/W /NKX2-5 eGFP/W hESC reporter line. We performed detailed electrophysiological, functional, and transcriptional studies to characterize the heart-field specificity of these hESC-derived CMs. Electrophysiological studies revealed that, despite the presence of atrial and ventricular action potentials (APs) in both FHF and SHF, there are significant differences in their AP duration and cycle length. In addition, both FHF and SHF CMs responded appropriately to adrenergic stimuli. Single-cell RNA sequencing analysis confirmed the absence of nodal genes within these populations and provided evidence of unique molecular signatures for isolating FHF- and SHF-like CMs. Finally, we identified CORIN as a novel cell surface marker for FHF CMs. Our studies provide a platform for investigating in vitro cardiovascular development, drug screening, and may facilitate a safe approach for cell therapy in heart disease.