Electrical stimulation of human embryonic stem cells: Cardiac differentiation and the generation of reactive oxygen species

Abstract

Exogenous electric fields have been implied in cardiac differentiation of mouse embryonic stem cells and the generation of reactive oxygen species (ROS). In this work, we explored the effects of electrical field stimulation on ROS generation and cardiogenesis in embryoid bodies (EBs) derived from human embryonic stem cells (hESC, line H13), using a custom-built electrical stimulation bioreactor. Electrical properties of the bioreactor system were characterized by electrochemical impedance spectroscopy (EIS) and analysis of electrical currents. The effects of the electrode material (stainless steel, titanium-nitride-coated titanium, titanium), length of stimulus (1 and 90 s) and age of EBs at the onset of electrical stimulation (4 and 8 days) were investigated with respect to ROS generation. The amplitude of the applied electrical field was 1 V/mm. The highest rate of ROS generation was observed for stainless steel electrodes, for signal duration of 90 s and for 4-day-old EBs. Notably, comparable ROS generation was achieved by incubation of EBs with 1 nM H₂O₂. Cardiac differentiation in these EBs was evidenced by spontaneous contractions, expression of troponin T and its sarcomeric organization. These results imply that electrical stimulation plays a role in cardiac differentiation of hESCs, through mechanisms associated with the intracellular generation of ROS.

Introduction

Heart disease and stroke, the principal components of cardiovascular disease, are the first and the third leading cause of death in the United States, accounting for nearly 40% of all deaths, more than all cancer combined [1]. Because cardiomyocytes are unable to regenerate following an injury in the adult heart, cell-based therapies provide a potential alternative approach to replace damaged myocardial tissue and restore cardiac function. A major limitation toward this goal is the lack of donor cells. Although human embryonic stem cells (hESCs) have enormous potential as a source of cardiovascular cells, the regulatory elements mediating their differentiation to cardiomyocytes are largely unknown [2]. The full therapeutic utility of hESC will only be realized when they may be predictably differentiated into a range of cell lines [3], [4], [5].

Numerous studies have demonstrated the importance of chemical and physical stimuli in directing stem cell differentiation [6], [7], [8], [9]. Endogenous electromagnetic fields (EF) are present in the developing and regenerating tissues, either in the extracellular space or in the cell cytoplasm, and they range in strength from a few mV/mm to several hundred of mV/mm [3], [4]. Combined with diffusible chemical gradients, EF lead to the polarization and the formation of spatial patterns in developing embryo [12], [13], [14] creating directional signals necessary for the proper placement of the components of the organism.

Exogenous EF applied in vitro have been shown to influence cell behavior [5]. Several studies have reported galvanotaxis in a variety of cultured cells after stimulation with a constant direct current (DC) field comparable with those detected in vivo [6]. The cell responds to an externally applied EF by the passive and active intracellular influx of ions, such as calcium and sodium [7], [8], or the localization of lipid and epithelial growth factor (EGF) receptors in the membrane [9].

Exposure of mouse embryonic stem cells (mESCs) to EF has been found to promote cardiomyogenic [10] and angiogenic [21], [22] differentiation. However, the course of action of electrical stimulation on the activation of cell differentiating pathways is poorly understood. One possible mechanism involves the generation of reactive oxygen species (ROS) within the cell [10], [11]. Sauer and colleagues [10] demonstrated that stimulation with an exogenous electric field increases intracellular ROS production in mouse embryoid bodies (EBs). ROS are highly reactive molecules generated during the normal metabolism of oxygen by NADPH oxidases or as side products of several enzymatic systems (e.g., cyclooxygenases, nitric oxide synthases, mitochondrial cytochromes). Although excessive concentration of ROS, such as superoxide anions (O₂⁻) and hydrogen peroxide (H₂O₂), is considered destructive and results in inhibition of gene expression [12], [13], small amounts of ROS function as intracellular second messengers and activate signaling cascades involved in growth and differentiation of many cell types [24], [26], [27], [28].

ROS have been implied in the regulation of cardiogenesis [14] via a variety of mechanisms. When cardiac cells are stimulated by cytokines [15], growth factors, hormones or mechanical stress [16], they elicit a small oxidative burst and generate low concentrations of ROS. During myocardial infarction, cardiac cells generate large amounts of free radicals and ROS, which are involved in the signaling and activation of the intrinsic repair mechanisms of the damaged myocardium [17], [18]. ROS activate the mitogen-activated protein kinase (MAPK) pathways in mouse EBs [19], enhancing angiogenesis via activation of ERK1,2 and JNK and cardiomyogenesis via phosphorylation of ERK1,2, JNK and p38. An NADPH oxidase-like enzyme is involved in the ROS-related cardiogenesis during mESC differentiation [2].

In previous studies done in our lab, a biomimetic system designed to deliver electric signals mimicking those in native heart resulted in the progressive development of conductive and contractile properties characteristic of cardiac tissue, including cell alignment and coupling, increased amplitude of synchronous construct contractions and a remarkable level of ultrastructural organization [32], [33]. In order to control cell behavior through the efficacious application of EF, we have also found it necessary to characterize the stimulation and understand its mechanisms of action at the biological level [26], [29], [30], [31]. Because electric charges are carried by ions in physiologic media and by electrons in electrodes and electric circuitry, at the electrode-medium interface, there is a transduction of charge carriers from electrons to ions.

Charge transfer can occur through three mechanisms: (i) non-Faradaic charging/discharging of the electrochemical double layer, (ii) reversible Faradaic reactions and (iii) non-reversible Faradaic reactions. The relative presence of each mechanism can be determined by electrochemical impedance spectroscopy (EIS), from which an equivalent circuit of the stimulation system can be constructed. Equivalent circuit modeling can provide insight into the behavior of the system being analyzed (which can be highly non-linear, depending on geometry, electrolyte composition, electrode composition and surface properties). In order to control cell behavior through the application of EF, we have characterized various electrode materials.

Taken together, these previous studies imply that electrical stimulation could be utilized to mediate hESC differentiation towards cardiac lineages and motivated the present study of the effects of electrical stimulation on the generation of ROS and cardiogenic differentiation of hESCs. The use of a simple yet highly controllable bioreactor system enabled us to probe in a systematic way the effects of multiple experimental variables (electrode material, duration of electrical stimulation, developmental stage of the cells). The conditions of stimulation were determined based on our cardiac tissue engineering studies [29], [30], [31] and previously reported effects of electrical signals on stem cell differentiation [10], [11]. The ROS generation and the expression of cardiac markers were correlated to each other and the experimental variables.