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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 ZonMW and the Dutch Heart foundation MKMD grant 114021512 and Dutch Heart Foundation Dekker fellowship 2020T023 Rationale: Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes represent an excellent in vitro platform to study cardiac development and model patient-specific diseases. However, their widespread application in drug discovery and regenerative approaches has not yet been realized at least in part due to limited comparative characterization of the available cardiomyocyte subtypes and lack of multicellular models that allow assessment of physiologically relevant parameters. Objective Perform comprehensive characterization of hiPSC-derived sinoatrial nodal cardiomyocytes (SANCM), atrial cardiomyocytes (ACM) and ventricular cardiomyocytes (VCM) from 2D and 3D cultures. Methods SANCM, ACM and VCM were generated using directed differentiation protocols. Electrophysiological analysis was performed by single cell patch-clamp. Subtype-specific differences were further characterized by response to neurohumoral agents noradrenaline, and carbachol and If blocker ivabradine. Next, we generated EHTs to evaluate the effect of 3D culturing on cardiomyocyte subtypes and performed optical mapping. To establish a model to study impulse initiation and propagation in vitro, we generated binary EHTs (BIN-EHTs) composed of heteropolar ends of SANCM and ACM. Results Gene expression analysis and single cell electrophysiology confirmed identities of respective cardiomyocyte subtypes. While response to noradrenaline and carbachol were as expected, ivabradine testing showed the functional presence of pacemaker current If in ACM, besides SANCM. After three weeks in culture as EHTs, cardiac structural genes were markedly upregulated in all groups. Optical mapping demonstrated a three-fold increase in conduction velocities in ACM and VCM while SANCM EHTs retained slower conduction velocities recapitulating in vivo differences. In BIN-EHTs, SANCM end of the EHT consistently paced the tissues under baseline conditions. Upon treatment with ivabradine, cycle length of BIN-EHTs increased and impulse initiation switched to ACM end in the majority of tissues. Conclusions We performed comprehensive characterization of hiPSC-cardiomyocyte subtypes, which recapitulated salient features of their in vivo counterparts. BIN-EHT constructs composed of SANCM and ACM are a valuable rudimentary model for investigating impulse formation and propagation in vitro. [ABSTRACT FROM AUTHOR]

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. Open in new tab Download slide [ABSTRACT FROM AUTHOR]