Directly Induced Neural Differentiation of Human Adipose-Derived Stem Cells Using Three-Dimensional Culture System of Conductive Microwell with Electrical Stimulation.

Adipose-derived stem cells (ADSCs) have the capacity to differentiate into neural precursor cells which can be used for nerve regeneration. However, their inherently low neurogenic differentiation efficiency limits further clinical applications. This study was designed to promote neurogenic differentiation efficacy of ADSCs by integrating conductive hydrogel-based microwells with electrical stimulation (ES). We hypothesize that ADSCs will differentiate more efficiently into neural precursor cells when electrically stimulated in conductive hydrogel microwells. To make the conductive hydrogel-based microwell, polyethylene glycol (PEG) diacrylate aqueous solution mixed with poly(3,4-ethylenedioxythiophene):polystyrene sulfonate (PEDOT:PSS) was patterned with the polydimethylsiloxane mold and exposed to UV light to induce photo-cross-linking of the conductive hydrogel. After seeding the ADSCs in the microwells, the cells formed distinct cell spheres in PEG microwells and wide disks in the PEG/PEDOT:PSS microwells. Although the microwells yielded varying three-dimensional (3D) cell aggregate structure, cell viability was not affected. After neurogenic differentiation with ES, the ADSC aggregates in PEG/PEDOT:PSS microwells with ES expressed greater positive neuronal differentiation markers compared to nonstimulated PEG/PEDOT:PSS microwells. Although all neuronal gene expression levels were greater in PEG microwells with ES, the increased rates of gene expression levels between treated and untreated PEG/PEDOT:PSS microwells were much higher compared to PEG microwells. This would mean that electrically stimulating ADSC aggregates in conductive microwells is an effective method in increasing neurogenic differentiation. Therefore, we propose a most effective strategy taking advantage of a 3D conductive culture system which can be useful in a wide variety of electrical application.