Enhanced Embryonic Stem Cell Differentiation to Cardiac Pacemaker Cells by Transduction with a Single Transcription Factor

Abstract

Background: Embryonic stem cells (ESCs) can spontaneously differentiate into heterogeneous aggregates of cardiomyocytes with atrial, ventricular, and pacemaker properties. We sought to bias the random cardiogenesis of ESCs toward pacemaker cardiomyocytes. Shox2 is an embryonic transcription factor essential for the patterning of pacemaker cells in the sinoatrial node (SAN). We hypothesized that Shox2 overexpression may tilt and dominate ESCs’ developmental program toward pacemaker myocytes.Methods: Mouse ESCs were transduced with an adenoviral vector expressing Shox2 or a control gene. Standard embryoid body (EB) method was used for differentiation.Pooled data are n≥3 with p 6-fold in Shox2-EBs compared to control at the early and late stages of differentiation. HCN4 protein level was up-regulated at all differentiation stages, with heightened cell surface expression in Shox2-EBs. Action potentials are mainly propagated by connexin(Cx)45 but not Cx43 in the SAN, which is mirrored in the atrial/ventricular myocardium. Shox2-overexpression led to an increase in the transcript (∼1.5-fold) and protein (∼3-fold) levels of Cx45. In contrast, Cx43 transcript and protein levels were reduced in Shox2-EBs by 33±5% and 30±10%, respectively, compared to control.Conclusion: Shox2 overexpression singularly biased ESC differentiation toward more pacemaker cells. Increased expression of NCX1, HCN4, Cx45, and down-regulation of Cx43 are all hallmarks of SA nodal cell biology. The data provide a novel and efficient platform to develop biological pacemakers from pluripotent cells.