Presentations

Abstract

Introduction: Induced pluripotent stem cells (iPSCs) are a widely utilized cellular model in regenerative medicine. Their ability to differentiate into any adult cell type makes them an extremely versatile tool for the development of cellular models for any tissue in the body. Currently, the standard methods for the derivation of differentiated cell types from iPSCs entail a combination of biomechanical and biochemical stimuli delivered in vitro. Our laboratory has previously shown that electro-stimulation of neural stem cells can be achieved in vitro via the delivery of pulsed electric fields in the picosecond range, resulting in metabolic and gene expression changes in the treated cells. Of particular interest was the upregulation of genes associated with terminal neural differentiation. The ability to stimulate stem cells in a non-contact manner, and without relying on chemically induced differentiation, could lead to novel neural and tissue engineering techniques. Here, we explored the effects of picosecond pulsed electric field delivery on induced pluripotent stem cells, and whether electro-stimulation of undifferentiated cells affects their lineage commitment.

Methods: A commercial 3D printer model, Felix 3.0 (FELIXrobotics, Ijsselstein, Netherlands), was adapted in our laboratory to enable the delivery of pulsed electric fields to biological cells in vitro, in a controlled and non-contact manner. The electrode configuration consisted of a coaxial cable transitioning into a pair of electrodes, which were consequently fixed to the head of the 3D printer. The picosecond pulses were generated by a 10 kV pulser (FID GmbH, Burbach, Germany) with a repetition rate of 1 kHz. Wild type pluripotent stem cells were previously generated in our laboratory from a human breast adipose tissue cell line. Cells were cultured and maintained in their undifferentiated state until treatment with pulsed electric fields at varying intensities: 20, 40 or 100 kV/cm. Following treatment, differential gene expression of control and treated cell populations was measured. Gene expression data were obtained through RNA extraction and purification, cDNA synthesis, and Real-Time quantitative PCR assays. Gene expression analysis was conducted using the 2-ΔΔCt of the average Ct for each subsample.

Results: Expression of all the pluripotency genes taken into consideration was upregulated following pulsing at 20kV/cm. A non-significant downregulation of the same target genes was observed when cells were pulsed at the higher intensities of 40kV and 100kV.

Conclusion: We observed a significant elevation of gene expression markers associated with pluripotency when 20kV pulses were applied, and a slight but not significant elevation at higher pulse intensities. Moreover, significant downregulation of germ layer markers, associated with stem cell differentiation, was observed when 40kV picosecond pulses were delivered. Finally, we showed that delivery of multiple pulses does not have a significant impact in the alteration of pluripotency markers expression. Overall, we show that picosecond pulse delivery has a protective effect and promotes the maintenance of pluripotency in iPSC colonies.