NODAL inhibition promotes differentiation of pacemaker-like cardiomyocytes from human induced pluripotent stem cells

Abstract

Directed cardiomyogenesis from human induced pluripotent stem cells (hiPSCs) has been greatly improved in the last decade but directed differentiation to pacemaking cardiomyocytes (CMs) remains incompletely understood. In this study, we demonstrated that inhibition of NODAL signaling by a specific NODAL inhibitor (SB431542) in the cardiac mesoderm differentiation stage downregulated PITX2c , a transcription factor that is known to inhibit the formation of the sinoatrial node in the left atrium during cardiac development. The resulting hiPSC-CMs were smaller in cell size, expressed higher pro-pacemaking transcription factors, TBX3 and TBX18 , and exhibited pacemaking-like electrophysiological characteristics compared to control hiPSC-CMs differentiated from established Wnt-based protocol. The pacemaker-like subtype increased up to 2.4-fold in hiPSC-CMs differentiated with the addition of SB431542 relative to the control. Hence, Nodal inhibition in the cardiac mesoderm stage promoted pacemaker-like CM differentiation from hiPSCs. Improving the yield of human pacemaker-like CMs is a critical first step in the development of functional human cell-based biopacemakers.

Bioengineering the Cardiac Conduction System: Advances in Cellular, Gene, and Tissue Engineering for Heart Rhythm Regeneration

“…It includes experimental tactics of embryoid body development (Yang et al, 2008 ) or monolayer attachment culture with specific biochemical conditioning (Laflamme et al, 2007 ). Yet, the differentiation of PSCs to cardiomyocytes mainly results in a mixture of atrial, ventricular, and nodal cells; therefore, the ultimate goal to guide the differentiation into a desired subtype and to create the biological pacemaker appears challenging ( Table 2 ) (Xu et al, 2002 ; He et al, 2003 ; Yang et al, 2008 ; Jung et al, 2012 , 2014 ; Mandel et al, 2012 ; Müller et al, 2012 ; Christoforou et al, 2013 ; Birket et al, 2015 ; Protze et al, 2016 ; Chauveau et al, 2017 ; Dorn et al, 2018 ; Zhang and Huang, 2019 ; Zhang et al, 2019a ; Yechikov et al, 2020 ).…”

“…Notably, only a few studies attempted to overcome this issue by focusing on deriving SAN-like cells from the cardiac differentiation and pacemaker specification of hiPSCs (Müller et al, 2012 ; Birket et al, 2015 ; Protze et al, 2016 ; Zhang and Huang, 2019 ; Yechikov et al, 2020 ). Calcium-activated potassium channels were demonstrated in hPSCs as a valid target to generate an enriched population of PC-like cells by conditioning with 1-ethyl-2-benzimidazolinone (1-EBIO).…”

“…More recently, Yechikov et al ( 2020 ) attempted to investigate the PC-like specification through Nodal inhibition. In the development of cardiac mesoderm, the inhibition of Nodal signaling downregulates a transcription factor, PITX2c, which represses SAN formation in the left atrium.…”

Application of Patient-Specific iPSCs for Modelling and Treatment of X-Linked Cardiomyopathies

“…The addition of specific molecules has been shown to promote or inhibit certain cardiomyocyte subtypes in order to generate more specific disease models and drug testing platforms [ 70 ]. Studies show that the induction of appropriate mesoderm with different concentrations of small molecules to activate or inhibit certain signalling pathways specifically yields atrial, ventricular, or pacemaker-type cardiomyocytes [ 71 , 72 , 73 , 74 , 75 , 76 , 77 , 78 ]. Cardiomyocyte differentiation kits are now commercially available.…”

Model Systems for Addressing Mechanism of Arrhythmogenesis in Cardiac Repair

“…An alternative strategy to reduce undesired cardiomyocyte subtype is direct differentiation to a specific subtype. Directed differentiation protocols that promote a higher fraction of ventricular, atrial, or pacemaking subtype have been reported [82][83][84][85][86][87][88]; however, none of the protocols could attain 100% yield of the desired subtype.…”