Validation of differentiated sinoatrial-like hiPSCs as a model of native sinus node myocytes

BackgroundHuman induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) constitute an attractive system for basic research and pharmacologic screening of new molecules of clinical interest. Numerous protocols aiming at differentiating atrial- or ventricular-like cardiomyocytes (hiPSC-CMs) are available. Conversely, only a few are available for obtaining patient-derived sinoatrial node-like pacemaker myocytes (PM-hiPSC-CMs). Here we validate a new protocol to differentiate mature PM-hiPSC-CMs as a model of native sinoatrial node (SAN) myocytes. MethodsWe generated PM-hiPSC-CMs through a 2D matrix-sandwich method promoting epithelial-to-mesenchymal transition and small molecule-based temporal modulation of Wnt signaling pathway. In addition, we treated our cells with triiodothyronine, dexamethasone and intracellular cyclic AMP (DTA) to enhance expression of proteins involved in intracellular Ca2+ handling. ResultsProteomic analyses showed expression of key SAN proteins in DTA-treated PM-hiPSC-CMs. Importantly, expression of proteins related to Ca2+ handling was increased in DTA-treated PM-hiPSC-CMs compared to untreated ones. DTA-treated PM-hiPSC-CMs displayed action potentials, ionic currents and intracellular Ca2+ dynamics typical of native SAN. In addition, pacemaker activity responded to both {beta}-adrenergic and muscarinic stimulation. ConclusionsOur data indicate that the differentiation protocol effectively generates PM-hiPSC-CMs with typical native human SAN features. This protocol may serve as a potential approach to generate PM-hiPSC-CMs from patients with history of sinoatrial node disfunction (SND) carrying different mutations in ion channels underlying pacemaking. In addition, these in vitro models of SND could be used for testing long-term vector-based gene therapeutic strategies to handle bradycardia.