Current-Controlled Electrical Point-Source Stimulation of Embryonic Stem Cells

Abstract

Stem cell therapy is emerging as a promising clinical approach for myocardial repair. However, the interactions between the graft and host, resulting in inconsistent levels of integration, remain largely unknown. In particular, the influence of electrical activity of the surrounding host tissue on graft differentiation and integration is poorly understood. In order to study this influence under controlled conditions, an in vitro system was developed. Electrical pacing of differentiating murine embryonic stem (ES) cells was performed at physiologically relevant levels through direct contact with microelectrodes, simulating the local activation resulting from contact with surrounding electroactive tissue. Cells stimulated with a charged balanced voltage-controlled current source for up to 4 days were analyzed for cardiac and ES cell gene expression using real-time PCR, immunofluorescent imaging, and genome microarray analysis. Results varied between ES cells from three progressive differentiation stages and stimulation amplitudes (nine conditions), indicating a high sensitivity to electrical pacing. Conditions that maximally encouraged cardiomyocyte differentiation were found with Day 7 EBs stimulated at 30 microA. The resulting gene expression included a sixfold increase in troponin-T and a twofold increase in beta-MHCwithout increasing ES cell proliferation marker Nanog. Subsequent genome microarray analysis revealed broad transcriptome changes after pacing. Concurrent to upregulation of mature gene programs including cardiovascular, neurological, and musculoskeletal systems is the apparent downregulation of important self-renewal and pluripotency genes. Overall, a robust system capable of long-term stimulation of ES cells is demonstrated, and specific conditions are outlined that most encourage cardiomyocyte differentiation.

FIGURE 1

Microelectrode arrays (MEA). Top: Micrograph of the MEA. An array of 6 × 6 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. Bottom: Image of an assembled microelectrode array chip, which is glued and wire-bonded to a printed circuit board carrier.

FIGURE 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 (i.e., electrodes and cell culture).

FIGURE 3

Dose–response curve of electrically stimulated ES cells. ES cells at three different differentiation stages were locally stimulated using the MEAs over three stimulation amplitudes. Early Stage ES cells were relatively unchanged following low and mid-level amplitude electrical stimulation, but were still able to significantly up-regulate β-MHC at the highest level of stimulation. Intermediate Stage ES cells demonstrated increased sensitivity, and while cardiomyocyte markers were increased up 30 µA, higher levels of stimulation may have the opposite effect, as seen by the decrease in β-MHC and troponin-T, and the increase in nanog. Stimulation at the Terminal Stage, on the other hand, showed an increase in both cardiomyocyte and stem cell markers. N = 6–8 per differentiation stage; *P < 0.05.

FIGURE 4

Partial stimulation of ES cells. To investigate the role of continuous stimulation, ES cells at the Intermediate Stage were stimulated for only half the typical 4-day period at 30 µA. ES cells that were stimulated for the first 2 days and then incubated for another 2 days were able to demonstrate the same amount of β-MHC as continuous 4-day stimulation. However, troponin levels were relatively unchanged, and the stem cell marker nanog levels were slightly higher. In the reverse situation where ES cells were not stimulated until the second half of the experiment, both cardiomyocyte cell markers did not show significant changes, while nanog levels were increased, although not significantly. N = 6–8 for each experimental condition; *P < 0.05.

FIGURE 5

Immunostaining of electrically stimulated cells. Following 4 days of continuous stimulation of Intermediate Stage ES cells at 30 µA, samples were fixed for fluorescent analysis. In both stimulated and control cases, cells appeared confluent, and were confirmed with nuclei staining (top two panels). In addition to that, cells were stained for troponin-T (lower two panels). Qualitatively, stimulated samples displayed higher amounts of troponin-T and is repeatedly observed in other samples (N = 4). The increase in troponin-T due to stimulation was not limited to the areas around the electrodes (highlighted in the dotted regions), but nonetheless stayed within the general vicinity.

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 in development, many specific physiological systems such as the nervous, hematological, musculoskeletal, and cardiovascular development were noted.