Electrical stimulation is increasingly being used to modulate human cell behaviour for biotechnological research and therapeutics. Electrically conductive polymers (CPs) such as polypyrrole (PPy) are amenable to in vitro and in vivo cell stimulation, being easy to synthesise with different counter ions (dopants) to augment biocompatibility and cell-effects. Extending our earlier work, which showed that CP-mediated electrical stimulation promotes human neural stem cell differentiation, here we report using electroactive PPy containing the anionic dopant dodecylbenzenesulfonate (DBS) to modulate the fate determination of human induced pluripotent stem cells (iPSCs). Remarkably, the stimulation without conventional chemical inducers resulted in the iPSCs differentiating to cells of the three germ lineages—endoderm, ectoderm, and mesoderm. The unstimulated iPSC controls remained undifferentiated. Phenotypic characterisation further showed a robust induction to neuronal fate with electrical stimulation, again without customary chemical inducers. Our findings add to the growing body of evidence supporting the use of electrical stimulation to augment stem cell differentiation, more specifically, pluripotent stem cell differentiation, and especially neuronal induction. Moreover, we have shown the versatility of electroactive PPy as a cell-compatible platform for advanced stem cell research and translation, including identifying novel mechanisms of fate regulation, tissue development, electroceuticals, and regenerative medicine.

Extended AbstractPolypyrrole (Ppy) is one of the most extensively studied conductive polymers (CPs) due to its ease of synthesis and surface modifications, high conductivity and its biocompatibility with various cell types [1]. Furthermore, the unique mechanical, electrical and chemical properties of CPs, which may be further enhanced with dopants, make these materials attractive for biomedical applications, such as tissue engineering, drug delivery and biosensors [2][3].The past decade has shown a resurgence of interest in electrical stimulation therapies, such as deep brain stimulation and transcranial direct current stimulation, for the treatment of schizophrenia (Sz). Sz is a debilitating, neurodevelopmental psychiatric disorder, affecting ~1% of the population worldwide, with high morbidity rate and no curative treatment for the Sz-induced cognitive deficits to date [4].One of the key pathologies of Sz are neural abnormalities, such as reduced neurite outgrowth in the brain [5]. Studies have shown that electrical stimulation, mediated by doped Ppy, promoted nerve cell (PC12) differentiation and increased neurite length and branching of mice primary neurons and human neural stem cells [6][7] [8]. Despite published effects of electrical stimulation, the comprehensive mechanism of electrical stimulation at the molecular level is yet to be elucidated. Furthermore, there are insufficient in vitro studies of electrical stimulation focused on the hypothalamus, a brain area affected by Sz. Therefore, this study aims to investigate the molecular effects of electrical stimulation, mediated by Ppy, on hypothalamic neurons and a hypothalamic model of Sz, in addition to the biocompatibility and efficacy of Ppy-mediated electrical stimulation.Polypyrrole/dodecylbenzene sulfonic acid (Ppy/DBS) films were prepared by galvanostatic electropolymerisation of aqueous pyrrole solution (0.2M), using an eDAQ potentiostat at a current density of 0.25 mA/cm 2 for 2 minutes with DBS (0.05M) as the dopant. A standard three-electrode electrochemical set-up, with gold-coated mylar as the working electrode, Ag|AgCl as the reference electrode and platinum wire mesh as the counter electrode; was used for the polymer growth. Four-well chambers were then glued onto individual Ppy/DBS film strips, and sterilized with 70% ethanol. Hypothalamic cells (mHypoA-59) were seeded into the chambers at a density of 0.5 x10 4 cells/well in DMEM, with 10% foetal bovine serum and 1% Penicillin-Streptomycin. Lids comprising platinum mesh electrodes were placed on top of the chambers with the electrodes touching the media. After 24 hours, the cells were electrically stimulated for 3 days using previously established parameters [7]. On day 4, the samples were fixed or collected for assay ology. Qualitative morphological analysis with immunocytochemistry showed increased neurite outgrowth of the electrically stimulated (ES)-cells compared to the non-ES controls. Quantitative real-time polymerase chain reaction (qRT-PCR) showed electrical stimulation signific...