Cardiac Differentiation of Embryonic Stem Cells with Point-Source Electrical Stimulation

Abstract

The use of pluripotent stem cells as a means to repair damaged heart tissue has recently emerged as a promising, yet controversial therapy. Despite the different approaches and the variety of cell types used, many of these procedures have been met with mixed success. The lack of understanding of the differentiation and integration process, notably with respect to electrical signaling, significantly hampers the development of these therapies. A system was thus developed allowing the use of point source electrical stimulation on embryonic stem (ES) cells to study the effect of physiologically-relevant electrical stimulus. When modulating the amplitude of the stimulus over various differentiation stages of embryonic stem cells, differences in the proportions of cardiomyocytes to embryonic stem cells were observed through quantitative PCR. The use of this technique might have larger applications in understanding molecular pathways towards the regeneration process.

I. Introduction

HEART failure is a devastating condition due primarily to the limited ability of the human heart to regenerate. Despite the success of many forms of therapy including implantable devices, whole organ transplants, and various pharmacological agents, many complications remain. Recently, a more biological approach using pluripotent stem cells to aid intrinsic repair mechanisms in damaged heart tissue is rapidly being explored. Cardiac grafting procedures use a variety of stem cell-derived cardiomyocytes and undifferentiated stem cells, but have been met with mixed success. While current evidence suggest that stem cell grafts are viable as well as physiologically functional [1,2], the mechanisms of the integration process remain largely unknown. Indeed, transplanted cells may contribute to a variety of events that lead to increased heart function, including differentiation into cardiomyocytes, angiogenesis, cell fusion, or paracrine effects. For heart tissue in particular, successful cell transplantation should take into account the role of electrical and mechanical coupling.

Electrical signals are present during normal fetal development [3], and it is thus reasonable to hypothesize that similar stimulus may play a role in the differentiation and integration of stem cells introduced into a cardiac tissue. Undifferentiated embryonic stem (ES) cells tend to spontaneously differentiate into multiple lineages, with approximately only five percent differentiating into cardiomyocytes. Furthermore, implanting ES cells in-vivo run the risk of teratoma formation [4] or induced arrhythmia [5]. Although many factors play a significant role in the differentiation of stem cells following implantation, the role of electrical stimulation from endogenous, in-vivo pacing is poorly understood.

Typical stimulation methods often rely on producing a homogenous electrical potential between two large electrodes over a small volume. It has been shown that such electrical field stimulation over an eight day period increased the amplitude of synchronous contractions in a tissue construct of cardiac cells, and promoted structure (presence of striations, ordered gap junctions) in otherwise disorganized cardiomyocytes cultured [6]. With mouse embryonic stem cells, the application of a single 90 second DC pulse over an embryoid body (EB) had in certain cases doubled the yield of beating EB’s [3].

Cells in the myocardium are depolarized by local currents, propagating in a wave-like pattern. Synchronous, field stimulation would thus not adequately mimic the electrical micro-environment stem cells may be subjected to in a graft. To address this issue, a local point-source stimulation approach is proposed, thought to reproduce more closely the boundary conditions of the graft, where only the peripheral cells in contact with the healthy myocardium would be subjected to local currents and their resulting fields. The system relies on the use of planar microelectrode array technology (MEA) with integrated recording and stimulation electrodes [7]. Stimulation electrodes provide localized current injection into the cell culture. The recording electrodes will allow the detection of nascent depolarization in differentiating cells, as well as the measurement of conduction properties of the differentiated tissue. Together with patterned co-culture techniques developed in our lab, this unique tool will allow the monitoring of the functional integration of the differentiated tissue into a primary tissue. In particular, the ability to evaluate the conduction mismatch will be key in order to assess the risk of arrhythmia development. This paper reports on the development of the stimulation system and the response of murine ES cells at different differentiation stages to a range of physiologically-relevant electrical stimulation parameters. This study is a first step toward the characterization of the functional integration of stem cells and stem cell-derived cardiomyocytes into a host tissue.

II. METHODS

A. Electrical Stimulation

The MEA’s used for this study contained stimulation electrodes symmetrically arranged across the surface with varying surface geometries (see Fig. 1). These MEAs have been previously described in [7]. All the outer stimulation electrodes were connected together and then used to stimulate the cells with the same signal. An MSP430 microcontroller (Texas Instruments, Dallas, TX) was used to control a 16-bit bipolar digital-to-analog converter (DAC), which was driving a modified Howland voltage-controlled current source (see Fig. 2). The desired waveform, rate, duration, and pulse amplitude were programmed for continuous and stable pacing. The type of stimulation applied was an anodic-first biphasic waveform with a duration of 10 msec. Pulses were applied at a frequency of 1 Hz (60 pulses per minute). The amplitude of the applied current was varied depending on the experiment between 10, 30, and 60 μA (8.3, 24.9, and 49.8 μA/mm², respectively). These values were chosen to be of similar order of magnitude to transmembrane currents estimated through a cell during a physiological action potential [8], although 30 μA (24.9 μA/mm²) may already be considered on the higher end. In order to prevent hydrolysis at the electrodes, the maximum total current across the electrode was kept under 65 μA, corresponding to a voltage under 0.9 V [9]. Voltages were monitored with a current sense circuit (see Fig. 2) to ensure compliance. Each sample was monitored to ensure the voltage did not exceed these limits. In each culture, a platinum wire was placed in the medium bath to serve as a ground electrode path for the injected current. Control samples were placed under the exact same conditions, but with no electrical stimulation applied.

Fig. 1.

Bottom: Image of an assembled microelectrode array chip, which is glued and wire-bonded to a printed circuit board carrier. Top: micrograph of the MEA. An array of 6x6 electrodes is located in the center used for electrical sensing. The larger electrodes on the periphery contained in the dashed boxes are used for stimulation [10].

Fig. 2.

Two operational amplifiers were configured as a voltage-controlled current source to deliver a precise current at each pulse. In addition, a high-side current sense was applied using an instrumentation amplifier to monitor the current delivered to the load (ie. electrodes and cell culture).

B. Cell Culture

Murine ES-D3 cell line (CRL-1934) was obtained from the American Type Culture Collection (ATCC; Manassas, VA). The ES cells were cultured to keep them in an undifferentiated, pluripotent state using 1000 IU/Ml leukemia inhibitory factor (LIF; Millipore, Billerica, MA), and grown over a layer of murine embryonic fibroblast feeder cells. The fibroblasts were inactivated using 10 μg/mL mitomycin C (Sigma-Aldrich, St. Lousi, MO). The surfaces of the culture dishes were coated with 0.1% gelatin. Cells were cultures in ES medium containing Dulbecco modified Eagle medium supplemented with 15% fetal calf serum, 0.1 mmol/L β-mercaptoethanol, 2 mmol/L glutamine, and 0.1 mmol/L nonessential amino acids as described previously [11]. The culture medium for the ES cells was changed on a daily basis, and cultures were passaged every one to two days.

To develop the ES cells into embryoid bodies, the “hanging drop” method was used as described previously [12]. LIF was withdrawn from the medium, and a cultivation of 400 cells were suspended in 18 μL hanging drops to form an aggregate of cells termed embryoid bodies (EB). At this point, the differentiation stage of the EB was noted as Day 0. After two days, each EB was transferred to its own well in an ultra-low attachment 96-well plate (Corning Life Sciences, Lowell, MA) for two days, after which they were further seeded onto 48-well plates.

At the specified differentiation stage, whole EB’s were dissociated with collagenase (Worthington, Lakewood, NJ) and cells were plated onto the MEA surface at a density of 32,000 cells/cm² (total of 80K cells). At least two hours prior to plating, the MEA surface was coated with Matrigel^™ (BD, Franklin Lakes, NJ). The cells were allowed to settle between 20 to 30 hours before electrical simulation was applied for four days. Medium was replaced every two days.

Embryoid bodies were cultured up to day 4, 7, and 12, and then plated onto the MEA surface. Stimulation was then applied at 10, 30, and 60 μA for four days, after which time the stem cells corresponded to three differentiation stages: Early, Intermediate, and Terminal [11],[13]. Gene expression analysis was performed at the end of the stimulation period for cardiac markers (β-MHC and troponin-T) [11] and ES cell markers (nanog) [14].

C. Real-Time PCR

After four days of stimulation, cells were harvested from the MEA using trypsin-EDTA (Invitrogen, Carlsbad, CA), and the RNA is extracted from the cells and is prepared as for reverse-transcriptase (RT) PCR. Gene expression of the stimulated stem cells were quantitatively measured using real time PCR (7900 HT; Applied Biosystems, Foster City, CA.). The four probes of interest were the cardiac cell markers β-MHC and troponin-T, the ES cell maker Nanog, and a housekeeping gene for control, gapdh. β-MHC, a ventricular specific protein, is typically regarded as an early stage marker. Troponin-T is essential for muscle contraction, and is regarded as a late stage marker. The numbers reported for the real-time PCR are the ratio of the relative expression of the gene of interest divided by the expression of gapdh.

D. Statistics

All data is presented as mean ± standard error of the mean (SEM). A Student’s t-test was performed between stimulated and non-stimulated control groups. Significance was considered at p < 0.05. Sample size in each stimulation group within each differentiation stage was between 6-8 samples.

III. Results

The system was used to evaluate the effect of localized electrical stimulation on the differentiation of embryonic stem cells at three differentiation stages.

ES cells stimulated at the Early Stage did not demonstrate much response at lower amplitude stimulus. However, stimulating at the highest amplitude of 60 μA yielded a statistically significant increase in the β-MHC levels. Otherwise, no significant changes were observed with troponin-T or nanog.

The situation changed when stimulating at the Intermediate Stage. A similar increase in β-MHC from the Early Stage was observed at 30 μA instead of 60 μA, and troponin-T displayed a six-fold increase in relative expression compared with non-stimulated samples. However, high amplitude stimulation may have had a detrimental effect on cardiac marker expression, with lower values at 60 μA. Interestingly, the ES cell marker nanog increased significantly.

Terminal Stage stimulation also showed a different picture from the rest. In general, cardiac markers expression increased as the stimulus amplitude increased, yielding up to a six-fold increase in troponin-T levels when stimulated at 60 μA. In this case, however, expression of the embryonic stem cell marker did show an increase with stimulation, although not statistically significant.

Phenotypically, cell cultures did not display significant differences in electrical activity by the end of the stimulation period, as recorded by electrodes and visual observation, between stimulated and control samples. However, such phenotypical changes may not be macroscopically apparent at this stage of the differentiation process.

IV. Conclusion

Embryonic stem cells at different differentiation stages of development displayed varying levels of sensitivity towards electrical stimulation. In general, later stages tended to have larger changes in both cardiac and embryonic gene expression. At the Terminal Stage, stem cells differentiating to cardiomyocytes showed a positive correlation to the stimulation amplitude. Interestingly, higher amplitude stimulation was not always correlated with increased expression, as demonstrated in stimulating at the Intermediate Stage. In any case, these results demonstrate the sensitivity of embryonic stem to their local electrical environment, and in particular to a stimulation pattern mimicking the endogenous physiological pacing [8].

The differences found in stimulated groups also raise the issue of spatial distribution of differentiated cells with respect to stimulation electrodes. Since the undifferentiated tissue is not capable of supporting the propagation of a depolarization wave, electrically-induced differentiation would occur only in the direct neighborhood of electrodes and fully-differentiated, electrically active cells. More wide-spread differentiation might point toward a joint paracrine signaling pathway. Further studies are required to examine any correlation between the number of cells physically attached to a stimulation electrode and the observed gene expression, or by direct immunochemical staining. Analogously, one might not expect all implanted stem cells to be directly stimulated electrically, and by simulating the physiological micro-environment, the importance of cell coupling (both electrical and chemical) can be revealed.

While differentiation is a first and necessary step toward functional repair, integration (participation in electrical conduction and mechanical contraction) is equally important. Future work using the presented system will use the full potential of MEAs to monitor in co-cultures the development of action potentials and propagation patterns (including conduction mismatches responsible for increased risks of arrhythmias), and study the impact of electrical stimulation on these parameters. If electrical stimulation appears beneficial, one might suggest the coordinated effort of stem cell implantation and cardiac pacing in the region of interest.

Fig. 3:

Expression of cardiac (β-MHC, troponin-T) and ES cell (nanog) markers after electrical stimulation at the Early Stage did not exhibit any sensitivity to stimulation until higher amplitude pulses were applied at 60 μA, where statistically significant up-regulation of β-MHC was observed. N=6-8 per group. * p<0.05, significantly different from control.

Fig. 4:

Electrical stimulation at the Intermediate Stage up-regulated both cardiac markers up to 30 μA. However, the trend is not continued with high amplitude pulses, as cardiac expression decreased. The expression of ES cell marker also significantly increased at high amplitude stimulation. N=6-8 per group. * p<0.05, significantly different from control.

Fig. 5:

Terminal Stage stem cells also reacted differently to electrical stimulation. In this case, higher amplitude stimulation led to higher cardiac marker expression. In addition, ES cells markers showed an increase due electrical stimulation, also not statistically significant. N= 6-8 per group. * p<0.05, significantly different from control.