Neuronal Differentiation of Human Embryonic Stem Cell-Derived Neural Crest Stem Cells by Pulsed Electrical Field

Abstract

This work surveyed the potential of using exogenous pulsed electrical field stimuli to guide the differentiation of human embryonic stem cell (hESC) derived-neural crest stem cells (NCSCs) towards their neuronal lineage. A vertical electrode bioreactor able to vary the parameters of direct current pulse frequency, width and intensity was fabricated for this purpose. Dendritic morphology was observed for hNCSCs cultured on cathodes subject to 1 Hz, 50 ms pulse at 150mV/mm and 200mV/mm for 24 hours. The morphological phenomena were observed in a dosage and polarity orientation dependent manner; however, significant apoptosis was observed post stimulation.

hNCSCs and hESCs were then cultured on cathodes and subject to 1Hz, 50ms pulse at 150mV/mm and 200mV/mm for 1.5 hours/day and 3 hours/day for 8 days. The expression of neuron-specific class III beta tubulin (Tuj1) was observed through immunofluorescence post stimulation and the expression levels of Tuj1 were qualitatively higher for the stimulated hNCSCs and hESCs as compared to the controls. Apoptosis was only observed in hNCSCs subject to the harshest condition.

Ethylene glycol tetraacetic acid (EGTA) was used to sequester extracellular Ca2+ in the media during the stimulation of hNCSCs on cathodes subject to 1Hz, 50ms pulse at 200mV/mm for 24 hours. As EGTA concentrations were increased from 0mM to 2mM, dendritic morphology was reduced. Thus, the morphological phenomena were correlated to the available free extracellular calcium concentrations. Further investigation into the intracellular calcium concentrations in stimulated hNCSCs and hESCs was accomplished through the Fluo-4 assay. hNCSCs and hESCs were stimulated on cathodes subject to 2Hz, 50ms pulse at 200mV/mm for 5 hours. Increased chronic levels of intracellular calcium were observed for hNCSC and hESC samples post stimulation.