Canonical Wnt signaling promotes pacemaker cell specification of cardiac mesodermal cells derived from mouse and human embryonic stem cells

Abstract

Cardiac differentiation of embryonic stem cells (ESCs) can give rise to de novo chamber cardiomyocytes and nodal pacemaker cells. Compared with our understanding of direct differentiation toward atrial and ventricular myocytes, the mechanisms for nodal pacemaker cell commitment are not well understood. Taking a cue from the prominence of canonical Wnt signaling during cardiac pacemaker tissue development in chick embryos, we asked if modulations of Wnt signaling influence cardiac progenitors to bifurcate to either chamber cardiomyocytes or pacemaker cells. Omitting an exogenous Wnt inhibitor, which is routinely added to maximize cardiac myocyte yield during differentiation of mouse and human ESCs, led to increased yield of spontaneously beating cardiomyocytes with action potential properties similar to those of native sinoatrial node pacemaker cells. The pacemaker phenotype was accompanied by enhanced expression of genes and gene products that mark nodal pacemaker cells such as Hcn4, Tbx18, Tbx3, and Shox2. Addition of exogenous Wnt3a ligand, which activates canonical Wnt/β‐catenin signaling, increased the yield of pacemaker‐like myocytes while reducing cTNT‐positive pan‐cardiac differentiation. Conversely, addition of inhibitors of Wnt/β‐catenin signaling led to increased chamber myocyte lineage development at the expense of pacemaker cell specification. The positive impact of canonical Wnt signaling on nodal pacemaker cell differentiation was evidenced in direct differentiation of two human ESC lines and human induced pluripotent stem cells. Our data identify the Wnt/β‐catenin pathway as a critical determinant of cardiac myocyte subtype commitment during ESC differentiation: endogenous Wnt signaling favors the pacemaker lineage, whereas its suppression promotes the chamber cardiomyocyte lineage.

Precise Correction of Heterozygous SHOX2 Mutations in hiPSCs Derived from Patients with Atrial Fibrillation via Genome Editing and Sib Selection

“…To prove the suitability of these lines as a cardiac disease model, patient-derived hiPSCs and their isogenic counterparts were differentiated into ventricular-, atrial-, and nodal-like cardiomyocytes using previously described protocols ( Foo et al., 2018 ; Liang et al., 2019 ; Zhang et al., 2015a ) ( Figures 6 A, 6E, and 6I). Flow-cytometry analysis of the pan-cardiac marker cardiac Troponin T showed comparable differentiation capacities between isogenic pairs ( Figures 6 B, 6F, and 6J).…”

“…Differentiation into ventricular- ( Foo et al., 2018 ), atrial- ( Zhang et al., 2015a ), and nodal-like cardiomyocytes ( Liang et al., 2019 ) was done as described previously. At day 20, differentiating myocytes were dissociated with papain (Worthington Biochemical Corporation) and analyzed for cTNT + cells by flow cytometry or further purified with the human PSC-derived Cardiomyocyte Isolation Kit (Miltenyi Biotech) followed by RNA isolation for qPCR analyses or plating on fibronectin-coated dishes for immunohistological analysis (detailed methods are described in the Supplemental Information).…”

Nanomaterials modulating stem cell behavior towards cardiovascular cell lineage

“…Furthermore, cardiac fibroblasts can transform into the myofibroblast with a contracting ability during myocardial infarction (reduction in blood flow to the heart). [28][29][30][31][32][33] Diseases that involve the myocardium are the most important clinical problems, which are the leading causes of death in developing countries. Coronary artery disease (CHD) or ischemic heart disease (IHD) is the most prevalent condition of the heart, which is the reduction of blood flow to the heart due to atherosclerosis.…”

Subtype-specific cardiomyocytes for precision medicine: Where are we now?

“…Precision production of subtype-specific cardiomyocytes has the potential to elucidate cardiovascular disease mechanisms and facilitate the development of individual-based therapies. For example, atrial-like cardiomyocytes can be used to model atrial disease for drug discovery, large-scale production of ventricular-like cardiomyocytes may be utilized as a cell therapy for myocardial infarction, and patient-derived pacemaker cells are promising source for biological pacemakers [82] Liang et al [88] Ren et al [89] Note: This summary is primarily based on the 2-D monolayer differentiation. RA can be added as early as day 3 for atrial CM differentiation in an EB-based differentiation protocol [18].…”