Conductive Polymer PEDOT:PSS-Based Platform for Embryonic Stem-Cell Differentiation

Abstract

Organic semiconductors are constantly gaining interest in regenerative medicine. Their tunable physico-chemical properties, including electrical conductivity, are very promising for the control of stem-cell differentiation. However, their use for combined material-based and electrical stimulation remains largely underexplored. Therefore, we carried out a study on whether a platform based on the conductive polymer poly(3,4-ethylenedioxythiophene):polystyrene sulfonate (PEDOT:PSS) can be beneficial to the differentiation of mouse embryonic stem cells (mESCs). The platform was prepared using the layout of a standard 24-well cell-culture plate. Polyethylene naphthalate foil served as the substrate for the preparation of interdigitated gold electrodes by physical vapor deposition. The PEDOT:PSS pattern was fabricated by precise screen printing over the gold electrodes. The PEDOT:PSS platform was able to produce higher electrical current with the pulsed-direct-current (DC) electrostimulation mode (1 Hz, 200 mV/mm, 100 ms pulse duration) compared to plain gold electrodes. There was a dominant capacitive component. In proof-of-concept experiments, mESCs were able to respond to such electrostimulation by membrane depolarization and elevation of cytosolic calcium. Further, the PEDOT:PSS platform was able to upregulate cardiomyogenesis and potentially inhibit early neurogenesis per se with minor contribution of electrostimulation. Hence, the present work highlights the large potential of PEDOT:PSS in regenerative medicine.

Keywords: PEDOT:PSS; conductive polymer; electrostimulation; embryonic stem cells; screen print.

Figure 1

Chronoamperometric characteristics of PEDOT:PSS and gold platforms. Platform wells were loaded with PBS buffer. In two-electrode mode, the electric current was determined under pulses (1 Hz, 200 mV/mm, pulse duration 100 ms). Right column shows magnified views. Charts represent typical data.

Figure 2

Depolarization of cytoplasmic membrane of mESCs under electrostimulation. EBs adhered to PEDOT:PSS platform wells (6 d) were loaded with voltage-sensitive probe DiBAC(4). The change in fluorescence upon stimulation with electric pulses (square wave, 1 Hz, 200 mV/mm, pulse duration 100 ms) recorded with a fluorescence microscope. Potassium chloride (40 mM) spike was used as a positive control. Records were processed in ImageJ software and normalized to the controls (0 mV/mm). The chart shows typical data from three replicates.

Figure 3

Elevation of cytosolic-calcium level under electrostimulation: EBs adhered to PEDOT:PSS platform wells (6 d) were loaded with calcium-sensitive probe Fluo-4 AM. The change in fluorescence upon stimulation with electric pulses (square wave, 1 Hz, 200 mV/mm, pulse duration 100 ms) recorded with a fluorescence microscope connected with camera. The record was processed in ImageJ software. (A) The chart shows typical data from three independent biological replicates. (B) Differences of fluorescence without electric stimulation and after stimulation for 15 min.

Figure 4

The effect of electrostimulation on expression cardiomyogenesis markers: Nkx2.5, Myh6, Myh7, Myl2, Myl7 on the PEDOT:PSS-based platform (left column) and the gold platform (right column). These genes represent markers of cardiomyocyte differentiation. The mRNA levels of NK2 transcription factor related locus 5 (Nkx2.5, (A)), myosin heavy chain 6 (Myh6, (B)), and myosin heavy chain 7 (Myh7, (C)), myosin light chain 2 (Myl2, (D)) and myosin light chain 7 (Myl7, (E)) were analyzed in mESC line R1 that was differentiated for 20 days. Different time points were studied (5 + 5, 5 + 10, 5 + 15 d). These time points represent individual phases of differentiation. EBs adhered to platforms were treated on day six with square electric pulses (1 Hz, 200 mV/mm, pulse duration 100 ms) for 15 min (PEDOT + EL, Au + EL). Culture plastics (Ctrl) served as the control, and a comparison to platforms without electrostimulation (PEDOT, Au) was drawn. Data are expressed as mean ± SEM (n ≥ 4). Differences between samples were analyzed by paired t-test and considered statistically significant for p < 0.05; they are marked with hashtags (#) for statistical significance to the control.

Figure 5

The effect of electrostimulation on expression of neural transcriptional factor Sox1 and LewisX antigen on PEDOT:PSS-based platform and platform with gold. These are markers of neural differentiation. The mRNA levels of transcription factor SOX-1 (A) were analyzed in mESCs line R1 which were differentiated for 20 days. Different time points were studied (5 + 5, 5 + 10, 5 + 15 d). These time points represent individual phases of differentiation. Level of antigen LewisX (B) was studied at the end-point (5 + 15 d). EBs adhered to platforms were treated on day 6 with square electric pulses (1 Hz, 200 mV/mm, pulse duration 100 ms) for 15 min (PEDOT + EL, Au + EL). Culture plastics (Ctrl) served as the control, and comparison to platforms without electrostimulation (PEDOT, Au) was drawn. Data are expressed as mean ± SEM (n ≥ 4). Differences between samples were analyzed by paired t-test and considered statistically significant for p < 0.05; they are marked with asterisks (*) for material vs. material + electrical stimulation and with hashtags (#) for statistically significance to the control.

Figure 6

(A) chemical formula of PEDOT:PSS (source: https://en.wikipedia.org/wiki/PEDOT:PSS (accessed on 12 December 2021)). (B) Schematic view of one well in a platform used for electrostimulation. Interdigitated electrodes (IDEs) covered with PEDOT:PSS are highlighted (C) The electrostimulation platform in the format of 24-well plate. (D) Image of a section of gold IDE covered with PEDOT:PSS showing good alignment of PEDOT:PSS with gold support. Image was taken with Nikon Eclipse E200 microscope equipped with D5000 camera (Nikon Europe BV, Amsterdam, The Netherlands).

Figure 7

Schematic illustration of the protocol for differentiation of mESCs. For the preparation of EBs from mESCs, the silicone microwells were used. After 24 h of incubation (Day 0), the EBs were transferred onto an agar-coated dish. On day 5 (Day 5), the EBs were seeded to the 24-well stimulation platform. On day 6 (Day 6*) cells were electrostimulated.