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.

Paucity of physiologically relevant cardiac models has limited the widespread application of human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes in drug development. Here, we performed comprehensive characterization of hiPSC-derived cardiomyocyte subtypes from 2D and 3D cultures and established a novel 3D model to study impulse initiation and propagation. Directed differentiation approaches were used to generate sinoatrial nodal (SANCM), atrial (ACM) and ventricular cardiomyocytes (VCM). Single cell RNA sequencing established that the protocols yield distinct cell populations in line with expected identities, which was also confirmed by electrophysiological characterization. In 3D EHT cultures of all subtypes, we observed prominent expression of stretch-responsive genes such as NPPA. Response to rate modulating drugs noradrenaline, carbachol and ivabradine were comparable in single cells and EHTs. Differences in the speed of impulse propagation between the subtypes were more pronounced in EHTs compared with 2D monolayers owing to a progressive increase in conduction velocities in atrial and ventricular cardiomyocytes, in line with a more mature phenotype. In a novel binary EHT model of pacemaker-atrial interface, the SANCM end of the tissue consistently paced the EHTs under baseline conditions, which was inhibited by ivabradine. Taken together, our data provide comprehensive insights into molecular and electrophysiological properties of hiPSC-derived cardiomyocyte subtypes, facilitating the creation of next generation composite cardiac models for drug discovery, disease modeling and cell-based regenerative therapies.

Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): European Research council starting grant 714866 and associated proof-of-concept grant 899422 Dutch Heart Foundation Dekker fellowship 2020T023 The sinoatrial node (SAN), the primary pacemaker of the heart generates electrical impulses that set the rhythm of the heartbeat. It is a multicomponent structure composed of "head", "tail" and "transitional" cardiomyocytes as well as proepicardium-derived mesenchymal cells. Previous studies have shown that pacemaker cells of the SAN originate from a common progenitor that also gives rise to the proepicardium. However, little is known of the molecular mechanisms that are implicated in 1) the divergence of pacemaker and proepicardial cells and, 2) the diversification of pacemaker cells into head, tail and transitional cells. Here, we performed single cell RNA sequencing (scRNA-seq) and trajectory inference analysis on human induced pluripotent stem cells (hiPSCs) differentiating to SAN-like cardiomyocytes (SANCM) to construct a roadmap of transcriptional changes and lineage decisions that guide these processes. scRNA-seq of differentiated cells revealed transcriptionally distinct cardiomyocyte populations that closely resemble different subpopulations of the in vivo SAN, such as "head", "tail" and "transitional" cells. Moreover, we identified a side population of proepicardial cells suggesting their shared ontogeny with SANCM, as also reported in vivo. Reconstruction of transcriptional trajectories revealed that the SANCM and proepicardial lineages diverge early on and that this lineage decision is determined by WNT signaling. Furthermore, we uncovered a role for TGFβ and WNT signaling in differentiation towards transitional and head SANCM subtypes, respectively. Manipulation of the identified signaling pathways enhanced gene expression patterns of the corresponding cell type, confirming their involvement in diversification towards SANCM subtypes. Our 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.