SHOX2 Overexpression Favors Differentiation of Embryonic Stem Cells into Cardiac Pacemaker Cells, Improving Biological Pacing Ability

• SHOX2 accentuates the molecular profile of pacemaker cells in differentiating ESCs
• SHOX2 increases the frequency and rate of spontaneously active cardiac derivatives
• SHOX2-overexpressing EBs function as biopacemakers when transplanted in vivo
• Wnt signaling underlies SHOX2-mediated pacemaker cell specification

SummaryWhen pluripotency factors are removed, embryonic stem cells (ESCs) undergo spontaneous differentiation, which, among other lineages, also gives rise to cardiac sublineages, including chamber cardiomyocytes and pacemaker cells. Such heterogeneity complicates the use of ESC-derived heart cells in therapeutic and diagnostic applications. We sought to direct ESCs to differentiate specifically into cardiac pacemaker cells by overexpressing a transcription factor critical for embryonic patterning of the native cardiac pacemaker (the sinoatrial node). Overexpression of SHOX2 during ESC differentiation upregulated the pacemaker gene program, resulting in enhanced automaticity in vitro and induced biological pacing upon transplantation in vivo. The accentuated automaticity is accompanied by temporally evolving changes in the effectors and regulators of Wnt signaling. Our findings provide a strategy for enriching the cardiac pacemaker cell population from ESCs.