Abstract
Each heartbeat is triggered by the sinoatrial node (SAN), the primary pacemaker of the heart. Studies in animal models have revealed that pacemaker cells share a common progenitor with the (pro)epicardium, and that the pacemaker cardiomyocytes further diversify into 'transitional', 'tail', and 'head' subtypes. However, the underlying molecular mechanisms, especially of human pacemaker cell development, are poorly understood. Here, we performed single cell RNA sequencing (scRNA-seq) and trajectory inference on human induced pluripotent stem cells (hiPSCs) differentiating to SAN-like cardiomyocytes (SANCMs) to construct a roadmap of transcriptional changes and lineage decisions. In differentiated SANCM, we identified distinct clusters that closely resemble different subpopulations of the in vivo SAN. Moreover, the presence of a side population of proepicardial cells suggested their shared ontogeny with SANCM, as also reported in vivo. Our results demonstrate that the divergence of SANCM and proepicardial lineages is determined by WNT signaling. Furthermore, we uncovered roles for TGFβ and WNT signaling in the branching of transitional and head SANCM subtypes, respectively. These findings provide new insights into the molecular processes involved in human pacemaker cell differentiation, opening new avenues for complex disease modeling in vitro and inform approaches for cell therapy-based regeneration of the SAN.