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.

Introduction

Mouse embryonic stem (ES) cells have been used as in vitro model to study cardiac development, to analyze toxic effects of drugs on cellular differentiation, and for human ES cells, as a potential source for cardiac repair [1], [2], [3], [4]. Cardiomyocytes with electrophysiological properties characteristic of pacemaker-, ventricular-, atrial-, His-purkinje- or sinus node-like cells have been generated from mouse [5], [6], [7] and human [8], [9], [10], [11] ES cells. A comparison of gene expression patterns and electrophysiological properties revealed that both mouse and human ES-derived cardiomyocytes are representative of early developmental stages [8], [12].

To enrich the number of ES cell-derived cardiomyocytes, strategies for directed cardiac differentiation have been established based on cardiac-specific promoters (e.g. α-MHC, MLC2v, cardiac α-actin) driving antibiotic resistance or reporter gene constructs [13], [14], [15], [16], [17], [18]. To enhance the efficiency of cardiac differentiation, growth factors and signaling molecules involved in heart development have been applied. Such cardiac-inducing factors are, for instance, signaling molecules of the bone morphogenetic protein (BMP), fibroblast growth factor (FGF) [19], [20], [21] and WNT [22], [23] families. These factors act in a spatiotemporal-dependent manner during mesoderm and cardiac development and initiate a cardiac-specific gene expression program, e.g. via the activation of Nkx2.5, GATA4/5/6 transcription factors and T-box (Brachyury) factors. Cytokines like cardiotrophin-1 [24], endothelin [25] and neuregulin-1 [26] also promote development of specific cell types of the cardiac conduction system. The vitamin A-derivative retinoic acid (RA) accelerates the differentiation of ES cells into the cardiac lineage and specifically induces ventricular cardiomyocytes [15].

Suramin, a symmetrical polysulphonated naphthylamine derivative of urea, is known for its anti-parasitic, anti-viral (HIV) and anti-cancer activities and exerts diverse biological effects on various cell functions including proliferation, migration and differentiation [27]. These activities have been attributed to its interaction with specific growth factors or cytokine receptors. Specifically, suramin prevents receptor binding of bFGF [28], hFGF-1 [29], EGF [30], IGF [31], PDGF [32], TGF-β [33], Wnt family members [34], IL1 [35], IL2 [36], IL4 [37] and TNF α [38]. In the case of hFGF-1, suramin directly binds to residues of the heparin and FGF receptor binding site and causes oligomerization of hFGF-1 [29], which results in the inhibition of FGF signaling. Besides the disturbance of growth factor-receptor binding, suramin has also been shown to affect growth factor signaling related to protein kinase C [39], [40], phosphatidylinositol kinase, diacylglycerol kinase [41], protein tyrosine phosphatases [42], or uncoupling of G-proteins [43]. In Xenopus embryos, suramin in a concentration-dependent manner alters the fate of the Spemann's organizer resulting in a shift of the dorso-ventral differentiation pattern and at high concentrations increased cardiac differentiation. These in vivo data led to our hypothesis that suramin would directly modulate cardiac differentiation of mouse embryonic stem (ES) cells.

Indeed, we found that high concentrations of suramin in a time-dependent manner induced formation of sinus node-like cells from differentiating ES cells. The process is concomitant with inhibition of neuronal, skeletal muscle and definitive endoderm (DE), but not mesendoderm cell differentiation. More specifically, suramin induced pleiotropic effects on transcripts of early mesoderm- and cardiac-specific genes, whereas at advanced stages, sinus node-specific genes, such as HCN4, Tbx3 and Tbx2 were significantly up-regulated. These data thus provide new insights into the process of ES-derived cardiac differentiation and specialization into sinus node-like cells. Because ES cell-derived cardiomyocytes can function as biological pacemakers [44], [45], these findings may foster the generation of patient-specific biopacemakers with therapeutic value for sick sinus syndrome and other bradycardiac disorders.