By utilizing current-controlled electrical stimulation for ES cells physically coupled to a microelectrode, a representative model was created to study the electrical effects on ES cells in the myocardium. One of the main advantages of using a current source was its ability to create a precisely controlled charged balanced waveform, minimizing electrochemical effects. Together with the microcontroller-based active grounding, this assured optimal performance for long-term stimulation, with safe and accurate delivery of current. This is to be contrasted with field stimulation systems, which typically require complex salt bridges to segregate cytotoxic byproducts generated at the electrodes and resulting from the much higher required voltages.
In continuous stimulation, embryonic stem cells at different differentiation stages of development displayed varying responses. In general, later stages tended to have larger changes in both cardiac and embryonic gene expression. At the Terminal Stage, stem cells exhibiting higher cardiac gene expression showed a positive correlation to the stimulation amplitude. However, higher amplitude stimulation was not always correlated with increased expression, as demonstrated in stimulating at the Intermediate Stage, where β-MHC and troponin-T were downregulated and nanog increased significantly. The increase in nanog levels was not expected, and it is possible that at this stimulation amplitude the ES cells remained in an embryonic state while control samples were differentiating. Such inflections of trends away from a neuronal and muscular lineage due to high amplitude stimulation were also observed in field stimulation studies.27
In any case, these results demonstrate the sensitivity of ES cells at various differentiation stages to their local electrical environment, and in particular to a stimulation pattern mimicking endogenous physiological pacing14
to yield statistically significant differences. Of particular interest is the fact that pacing Intermediate Stage cells at 30 µA can possibly increase the troponin-T expression by up to sixfold. Unlike field stimulation studies that uniformly depolarize whole volumes, simulating the physiological micro-environment by stimulating from a point-source reveals the importance of cell coupling (both electrical and chemical). Although quantitatively observing increases in the cardiac gene expression of electrically stimulated cells does not necessarily imply cardiac differentiation, it does give special insight into how ES cells first respond to their new environment. This work especially highlights their sensitivity, and suggests greater changes as the ES cells develop. Nonetheless, direct assessment of differentiation was seen through immunostaining, where cardiac markers appeared about three and a half times as prevalent compared to nonstimulated samples.
Furthermore, in addition to understanding the sensitivity of stimulated cells, the temporal patterns of stimulation appear vital. Continuous pacing of ES cells during the 4-day experiments was a requisite to promote the levels of differentiation observed. When ES cells were stimulated for 2 days and then left alone for an additional 2 days, the cardiac markers were only a fraction of the levels when continuously paced for 4 days. When the ES cells were paced for only the last 2 days of the 4-day period, cardiac markers were relatively unchanged while nanog levels increased by almost four times. A progression was found that started with an upregulation of nanog followed by β-MHC, and then troponin-T due to pacing.
The differences found in stimulated groups have raised 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. By fluorescently labeling a cardiac marker following stimulation, the distribution of the cardiac expression did not appear specifically confined to the stimulation electrodes, although did remain in the general vicinity. Although the concentration of cardiac marker troponin-T was higher over stimulation electrodes compared to the total average, it was not a very large difference. In addition to the theory of simply increased differentiation is the idea that cell–cell contact density, proliferation, and distribution over the stimulation electrode is altered within the stimulation period, and will be the subject of future investigations. So far, the direct role of electrical pacing does appear to play a part in differentiation, but this result points toward a joint paracrine signaling pathway, where electrical stimulation locally triggers the release of markers which may then promote cardiac differentiation throughout the culture. Although the stimulation conditions are different, this hypothesis aligns well with experiments done by Sauer et al.
which proposed the idea that reactive oxygen species act as secondary messengers as a mechanism to promote cardiac differentiation with application of a brief DC voltage field across the entire culture.
Of particular interest too is the fact that while cardiac differentiation may be triggered by electrical stimulation, this stimulation promotes differentiation into other tissues. This may help explain why statistical significance was not always observed in the real-time PCR analysis. In some field stimulation techniques on ES cells which affect the entire culture rather than only localized areas, major cardiac differentiation was not always elicited,27
but have seen a greater activation of neural pathways instead. In order to achieve a broader understanding of the effects of electrical stimulation, whole genome microarray analysis was performed. Simultaneous to the mild upregulation of mature gene programs is the apparent downregulation of important self-renewal and pluripotency genes, including Oct4 and Foxd3. The alteration in developmental gene programs of electrically stimulated mESCs is clearly seen with gene ontology (GO) overrepresentation analysis, which categorizes genes based on their annotations into functional groups (Supplemental Table 2
, see Supplemental Text Files 2
for full lists). The overrepresented GO terms in stimulated ES cells reveal a number of processes related to embryonic development, pattern specification, and tissue and organ development and morphogenesis. In contrast, dowregulated GO processes included cell organization and biogenesis, RNA and DNA metabolism, cell cycle and cytoskeletal organization, and microtubule biogenesis. Ingenuity Pathway Analysis (Ingenuity Systems Inc, Redwood City, CA) also demonstrates significant changes in developmental processes after electrical stimulation (, see also Supplemental Table 3). Based on Ingenuity’s database of gene networks, the functional categories that are most significantly changed by electrical stimulation include gene expression, organismal and organ development, cellular development, cell cycle, and specific physiological systems such as cardiovascular, neurological, hematological, and musculoskeletal development. Notably, the cardiovascular, neurological, and musculoskeletal systems are all electrically excitably tissues. Strangely, neither troponin-T nor β-MHC appeared on the microarray gene list, but that may only be because it did not reach statistical significance. Microarray analysis can often have issues with sensitivity, and this does not negate the real time PCR results but simply means the microarray cannot draw accurate conclusions about these specific genes.
FIGURE 6 Categories of altered gene expression following electrical stimulation. Ingenuity Pathway Analysis displays the function categories that exhibited the highest level of statistically significant changes following electrical stimulation. Among many changes (more ...)
Overall, this study describes a novel system used to electrically stimulate ES cells safely over long periods of time, and is capable of demonstrating subsequent changes in differentiation. Although some of the results were not statistically significant, it may be due to the limited area that the stimulation electrodes cover relative to the total culture area. Originally conceived as a feature to probe the spatial effects of stimulation, the particular size and configuration of the stimulation electrodes may not be optimal for differentiation. Another similar limitation of the system is the configuration of the recording electrodes in the center of the culture. The area that it covers (~4%) was unable to properly map any differences in the electrical activity between stimulated and nonstimulated samples. Differences were nonetheless quantified through real-time PCR, immunofluorescence, and whole genome microarray analysis.
While understanding the gene expression and subsequent differentiation is a first and necessary step toward functional repair, understanding integration with host tissue (participation to electrical conduction and mechanical contraction) is also important. Additional future work using the presented system will use the full potential of MEAs to monitor the development of action potentials and propagation patterns (including conduction mismatches responsible for increased risks of arrhythmias) in co-cultures, and study the impact of electrical stimulation on these parameters. Especially given the transient effects observed in these pacing experiments, continuous pacing may be necessary and one might even suggest the coordinated effort of stem cell implantation and cardiac pacing in the region of interest.