Differentiation induction of mouse embryonic stem cells into sinus node-like cells by suramin

Abstract

Background Embryonic stem (ES) cells differentiate into cardiac phenotypes representing early pacemaker-, atrial-, ventricular-, and sinus node-like cells, however, ES-derived specification into sinus nodal cells is not yet known. By using the naphthylamine derivative of urea, suramin, we were able to follow the process of cardiac specialization into sinus node-like cells. Methods Differentiating mouse ES cells were treated with suramin (500 μM) from day 5 to 7 of embryoid body formation, and cells were analysed for their differentiation potential via morphological analysis, flow cytometry, RT-PCR, immunohistochemistry and patch clamp analysis. Results Application of suramin resulted in an increased number of cardiac cells, but inhibition of neuronal, skeletal muscle and definitive endoderm differentiation. Immediately after suramin treatment, a decreased mesendoderm differentiation was found. Brachyury, FGF10, Wnt8 and Wnt3a transcript levels were significantly down-regulated, followed by a decrease in mesoderm- and cardiac progenitor-specific markers BMP2, GATA4/5, Wnt11, Isl1, Nkx2.5 and Tbx5 immediately after removal of the substance. With continued differentiation, a significant up-regulation of Brachyury, FGF10 and GATA5 transcript levels was observed, whereas Nkx2.5, Isl1, Tbx5, BMP2 and Wnt11 levels were normalized to control levels. At advanced differentiation stages, sinus node-specific HCN4, Tbx2 and Tbx3 transcript levels were significantly up-regulated. Immunofluorescence and patch-clamp analysis confirmed the increased number of sinus node-like cells, and electrophysiological analysis revealed a lower number of atrial- and ventricular-like cardiomyocytes following suramin treatment. Conclusion We conclude that the interference of suramin with the cardiac differentiation process modified mesoderm- and cardiac-specific gene expression resulting in enhanced formation of sinus node-like cells.

Embryonic Stem Cell–Derived CD166 + Precursors Develop Into Fully Functional Sinoatrial-Like Cells

“…5,50 In addition to the gene expression pattern, our CD166-selected cardiomyocytes also display the functional properties peculiar to the SAN cardiomyocytes. The main function of SAN myocytes is to spontaneously generate action potentials and to modulate cardiac rhythm to account for changes in oxygen demand.…”

Baicalin Maintains Late-Stage Functional Cardiomyocytes in Embryoid Bodies Derived from Murine Embryonic Stem Cells

“…Murine pluripotent stem cells yield a heterogeneous population of CMs [16,30]. Ventricular myocytes and atrial myocytes are morphologically, molecularly, and functionally distinct from pacemaker cells [31].…”

Stem Cell–Derived Nodal-Like Cardiomyocytes as a Novel Pharmacologic Tool: Insights from Sinoatrial Node Development and Function

“…Finally, Wiese et al (2011) tested on differentiating ESCs the effect of suramin, a compound known to interact with specific growth factors and cytokine receptors and, at high concentrations, to induce differentiation of heart structures in Xenopus embryos (Grunz, 1992). Transient treatment with 0.5 mM suramin from day 5 to day 7 during EB differentiation increased the yield of sinoatriallike cells that, as assessed by electrophysiologic analysis of action potential waveform, constituted between 50% and 70% of the total cardiomyocytes.…”

“…In agreement with functional data, suramin treatment doubled the expression levels of HCN4 and Tbx3 two well known markers of sinus node cells. At the same time, suramin prevented the differentiation of mESC toward the neuronal and skeletal muscle phenotypes (Wiese et al, 2011). …”