Enrichment differentiation of human induced pluripotent stem cells into sinoatrial node-like cells by combined modulation of BMP, FGF, and RA signaling pathways.

Biological pacemakers derived from pluripotent stem cell (PSC) have been considered as a potential therapeutic surrogate for sick sinus syndrome. So it is essential to develop highly efficient strategies for enrichment of sinoatrial node-like cells (SANLCs) as seed cells for biological pacemakers. It has been reported that BMP, FGF, and RA signaling pathways are involved in specification of different cardiomyocyte subtypes, pacemaker, ventricular, and atrial cells. We aimed to investigate whether combined modulation of BMP, FGF, and RA signaling pathways could enrich the differentiation of SANLC from human pluripotent stem cell (hiPSC). During the differentiation process from human induced pluripotent stem cell to cardiomyocyte through small molecule-based temporal modulation of the Wnt signaling pathway, signaling of BMP, FGF, and RA was manipulated at cardiac mesoderm stage. qRT-PCR, immunofluorescence, flow cytometry, and whole cell patch clamp were used to identify the SANLC. qRT-PCR results showed that manipulating each one of bone morphogenetic protein (BMP), fibroblast growth factor (FGF), and retinoid acid (RA) signaling was effective for the upregulation of SANLC markers. Moreover, combined modulation of these three pathways displayed the best efficiency for the expression of SANLC markers, which was further confirmed at protein level using immunofluorescence and flow cytometry. Finally, the electrophysiological characteristics of upregulated SANLC were verified by patch clamp method. An efficient transgene-independent differentiation protocol for generating SANLC from hiPSC was developed, in which combined modulating BMP, FGF, and RA signaling at cardiac mesoderm stage generates SANLC at high efficiency. This may serve as a potential approach for biological pacemaker construction.