Embryonic stem cells (ESCs) are pluripotent cells derived from the inner cell mass of blastocyst-stage embryos. Other types of stem cells (SCs) are isolated or induced from adult tissues. All SCs have unique regenerative abilities with a high potential for application in medicine [1
]. A plethora of approaches to direct ESCs into a particular cell lineage has been developed for in vitro use. The differentiation protocol depends on the origin of the cells and the intended direction of SC differentiation. The methods to regulate differentiation could be divided into three groups: biological, chemical and physical [3
]. Some growth factors and cytokines are able to stimulate ESCs and accelerate their differentiation. These are, for example, the TGF-beta family of proteins, insulin-like growth factor-1, leukemia inhibitory factor and cardiotrophin-1 [4
]. Further, some chemicals can stimulate differentiation as well. These include dimethyl sulfoxide, 5-Azacytidin or ascorbic acid, as well as exogenous free radicals and reactive oxygen species [3
]. The last group of stimulators is physical stimuli. This group includes mechanical forces, heat treatment and electrostimulation [3
]. The latter mentioned can affect endogenous electrical fields, which are essential for maintaining cellular homeostasis and are involved in many biological events [3
]. With the development of new conductive materials, electrostimulation is entering into the focus of basic as well as applied research.
Indeed, electrostimulation can be beneficial in tissue formation, tissue regeneration and wound healing, directed cell migration and alignment [6
]. There has been a special interest in myocardial regeneration after heart failure and cardiac-related diseases since the mammalian heart has a very limited self-regeneration capacity [7
]. In particular, cardiac regenerative medicine requires mature and well-defined cells in order to treat pathologies such as ischemic heart disease, cardiomyopathy and congenital cardiac birth defects in children [8
]. Electrical stimulation can potentially be one way to achieve this goal [10
]. In addition to enhanced cardiomyocyte formation, electrostimulation can result in the functional improvement of phenotypes (contractility, synchrony of beating) of formed cardiomyocytes even if prepared ex vivo [6
The electrostimulation of ESCs requires a platform to support the cells and to allow the application of external electrical fields. The vast majority of such platforms was constructed using inorganic materials. Indeed, the first systems for ESCs were made from platinum or stainless-steel electrodes in direct contact with the culture medium [10
]. More sophisticated systems used salt bridges to separate electrode buffers from the culture medium. Such an arrangement eliminated any adverse effect of the electrolysis of the culture medium, which stressed the cells to a high extent [14
The recent extensive development of organic semiconductors has rendered them to be used in biological applications as well. Among them, the conductive polymer poly(3,4-ethylenedioxythiophene):polystyrene sulfonate (PEDOT:PSS) has attracted a great deal of attention. Although the polymer chain of PEDOT shows conjugation of double bonds, the major contribution to electrical conductivity results from its nanocrystalline structure with extensive stacking. Hence, PEDOT:PSS requires post-deposition treatment to ensure favorable electrical properties [14
]. Due to its stability and biocompatibility, it has been suggested for biological applications. Indeed, short (a few hours) and mid-term (a few days) proof-of-concept applications have been implemented [15
]. Long-term applications have been rather scarce despite the fact that PEDOT:PSS can maintain its performance if processed appropriately [16
]. In connection with SCs, nanostructured PEDOT:PSS has been shown to manipulate cell adhesion. However, there is no clear general relationship between SC fate and PEDOT:PSS structure [18
]. Further, PEDOT:PSS-based composite materials have been introduced in order to provide 3D niches for SCs. In connection with electrostimulation, they appear to have great potential in regenerative medicine as shown by their support of the development of neurons from neural SCs [18
]. Other applications include osteogenic differentiation [22
]. Importantly, some works have indicated that PEDOT:PSS-based material could promote SC differentiation by itself [21
]. PEDOT:PSS was specifically used to promote cardiomyogenesis. The electrostimulation of mESCs with PEDOT:PSS-based electrodes resulted in the synchronous beating of clusters of cardiomyocytes [28
]. Further, recent work of Roshanbinfar et al., 2018 [27
] indicated that PEDOT:PSS-based hydrogel can promote cardiomyogenesis per se. The authors determined electrical conductivity to be the key player in the observed phenomenon. However, the use of organic semiconductors, including PEDOT:PSS, for a combined material-based and electrical stimulation to control SC differentiation, such as in the work of Yoshida et al., 2019 [28
], remains largely underexplored. By applying such an approach, the potential of organic conductive polymers and electrostimulation for regenerative medicine could be maximized.
This paper describes the construction of a PEDOT:PSS-based platform for the laboratory-scale electrostimulation of cells. The design took advantage of the precise screen printing of interdigitated PEDOT:PSS electrodes on a gold support. The platform was characterized in terms of biocompatibility and ability to produce electrical current at a pulsed-DC mode of operation. The impact of electrostimulation as well as the platform itself on mESC differentiation was determined based on lineage-specific gene and protein expressions. To our knowledge it is the first time that such a PEDOT:PSS-based design was used in a device for the electrostimulation of SCs.
The use of novel materials and electrostimulation to promote and control stem-cell differentiation is constantly gaining interest as a promising tool for regenerative medicine. The selective differentiation into a specific line is of special importance. [6
]. Further, organic conductive materials appear to be an excellent alternative to commonly used inorganic materials (e.g., gold or platinum) [19
]. Therefore, we determined if a PEDOT:PSS-based platform can be beneficial to the differentiation of mESCs compared to a gold-based platform.
The PEDOT:PSS platform was prepared using PEDOT:PSS paste deposited onto gold electrodes by screen printing. Such a method resulted in good quality and reasonably thin film, similar to the capabilities of spin coating. The major advantage was the precise deposition of PEDOT:PSS onto the supporting electrodes. Such a procedure in principle enables the production of cost-effective bio-electronic devices with submillimeter electrode size in a scalable manner. Though PEDOT:PSS in general reaches conductivity up to a few thousand Siemens, the thin layer (200 nm) in principle did not limit the overall conductivity of the platform. Importantly, the thin film was stabilized by ethylene-glycol treatment combined with heat annealing. This procedure improved the mechanical and electrical stability. Indeed, layers of PEDOT:PSS processed with ethylene glycol and thermal annealing were shown to be stable in terms of electrical conductivity for more than ten days [16
]. More recent research in this field indicated that PEDOT:PSS-coated gold electrodes could be stable in physiological media for up to four months [17
As demonstrated by the wetting angles, none of the materials in direct contact with cells (PEN foil, gold, PEDOT:PSS) showed excessively high hydrophobicity, which could prevent cell adhesion [29
]. In order to yield the maximum performance from the platforms, additional protein coating was carried out. Our previous work indicated that coating of PEDOT:PSS with collagen IV can provide excellent biocompatibility to cell cultures [16
]. Indeed, the present work supported this idea. The differentiation of ESCs into functional cardiomyocytes is a sensitive process that is only successful in very good culture conditions [31
]. In our work, the EBs resided on the platforms for 15 days. At the end of the differentiation, we regarded them as spread EBs (with clusters of cardiomyocytes). The ability of the PEDOT:PSS platform to enable the formation of beating loci with comparable beating frequency to the culture-plastics control proved the excellent biocompatibility of the PEDOT:PSS for long-term biological applications if coated appropriately.
Due to uncertainty about the possible combined effect of PEDOT:PSS and electrostimulation, the square DC electric pulses (1 Hz, 200 mV/mm, pulse duration 100 ms) based on the work of Hernandez et al. in 2016 [33
] were applied in the two-electrode mode. In the literature, there is a range from 60 mV/mm to 750 mV/mm that is covered for various type of cells [33
]. The voltage used in the present study corresponded to magnitude of endogenous physiological DC electrical fields that occur in animal tissues (10–200 mV/mm) [39
]. Further, such electrostimulation produced a discernable biological effect, speeding up the cardiomyocyte-beating onset during the differentiation (see Section 2.2
) The results clearly showed about 35 times higher capacitive current mediated by the PEDOT:PSS-covered electrodes compared with the gold electrodes. This generally corresponds with the ability of PEDOT:PSS to form high-electrical-capacity bio-interfaces [40
]. The faradaic currents were about six times higher for PEDOT:PSS; hence, such material produced a one-order-of-magnitude-higher ratio of capacitive to faradaic current. The faradaic current can be described by the electrocatalytic properties of PEDOT [41
] towards oxygen reduction [42
]. The essential component of electrostimulation on both platforms was capacitive current compared to the relatively low faradaic one. However, it cannot be excluded that a minor electrocatalytic oxygen reduction could affect cells in further experiments. Additionally, a PEDOT:PSS-based platform can produce less reactive oxygen species due to the oxygen reduction per electrical current in a pulsed mode of electrostimulation.
First, a proof-of-concept experiment with the PEDOT:PSS platform was carried out in order to determine if ESCs can produce an early response to the selected mode of electrostimulation. Our data showed that these cells could sense the stimulation as they responded with changes in membrane potential and an increase in cytosolic calcium. Such a finding is in accordance with the fact that even very short electrostimulation can have an effect on cell differentiation [33
], and we point to an early mechanism dependent on membrane depolarization behind such a response. Further, the data on the cytosolic-calcium level indicate that a longer electrostimulation has the potential to elicit a more pronounced response, which could be reflected in more intense late events. Indeed, the 15 min-long electrostimulation caused beating loci to occur about 1–2 days earlier compared to culture plastics and the platform without electrostimulation.
For differentiation, a general protocol was used. This approach did not induce any specific direction of stem-cell differentiation [43
]. Hence, it provided a sensitive tool to observe if the electrostimulation of the PEDOT:PSS and gold platforms, or the platforms themselves, can modulate possible directions of differentiation. As a model system, we chose a fine balance between differentiation of ESCs to cardiomyocytes and neural cells [45
]. Thus, for cardiomyogenesis the expression of transcription factor Nkx2.5 and the structural genes encoding the components of the contractile apparatus—Myh6, Myh7, Myl2, and Myl7—were chosen. The early neurogenesis was judged based on the expressions of Sox1 and Nestin in combination with the level of antigen LewisX. Additionally, expressions of Pax6, Mash1 and Tuj1 were determined to characterize later stages of neurogenesis [44
There was a marginal trend towards elevated cardiomyogenesis due to electrostimulation regardless of the platform type, as demonstrated by the Nkx2.5 expression. This was in accordance with the earlier onset of beating after electrostimulation (see above). The expression of genes Myh6, Myh7 and Myl7 was elevated on the PEDOT:PSS platform regardless of electrostimulation. The absence of such an effect on the gold platforms strongly indicated the predominant impact of the PEDOT:PSS material to boost cardiomyogenesis. Further, it implied no effect of the gold platform itself on cardiomyogenesis.
The expression of the contractile-apparatus genes was normalized to the level of the Nkx2.5 expression, which is relatively stable in cardiac precursor cells and cardiomyocytes. An increase in the ratio of contractile-apparatus-gene expression to the level of Nkx2.5 expression could point to a higher maturation of cells. The ratios of Myh6 to Myh7 and Myl2 to Myl7 expressions could provide information on the specific cardiomyocyte line (i.e., atrial or ventricular) [46
]. The inconsistencies and absence of differences among the listed ratios indicated no enhancement in cardiomyocyte maturation and no specific direction towards a particular type of cardiomyocytes.
The early neurogenesis was potentially reduced on the PEDOT:PSS platform. This was even more pronounced by electrostimulation on this platform, as indicated by Sox1 and the LewisX antigen. There was a potential reduction of early neurogenesis by the gold platform, as indicated by Sox1, Nestin and LewisX expressions, which could be reverted by electrostimulation. Further, the gold platform without electrostimulation showed a potential enhancement of neural maturation, as deduced from the elevated expression of Mash1. Such surprising properties of the gold platform were not documented in the literature and deserve a more detailed study beyond the scope of the present work.
Compared to the work of Hernández and similar papers, which showed a clear shift to cardiomyogenesis, the electrostimulation using a gold-electrode system in the present study only induced a marginal shift to this direction of differentiation [10
]. This was improved by the use of the PEDOT:PSS-based platform, but the effect of the material was more dominant. The combination of the improvement of cardiac differentiation by PEDOT:PSS with electrostimulation can be useful since the electrostimulation component can contribute to the functional improvement of the phenotype of formed cardiomyocytes, even if prepared ex vivo [6
As demonstrated by recent works, almost all research using the PEDOT:PSS or PEDOT:PSS-based materials to electrostimulate SCs was focused on the differentiation of neural SCs or used conditions supporting neural differentiation [18
]. In spite of this, fragments of the knowledge could be compared. Thus, a 10 min-long electrostimulation using a PEDOT:PSS-based scaffold was efficient in the upregulation of neural-cell maturation [24
], confirming our finding that even a short electrostimulation of SCs can affect late events. Our findings of the effect of PEDOT:PSS per se corresponded to the literature, as PEDOT:PSS-based material has rather inhibited neurogenesis without electrostimulation [20
The finding that PEDOT:PSS per se can significantly affect the ES differentiation could be hypothetically linked to the conductivity of the material or its surface properties. However, our data exclude the general electrical conductivity behind the effect of PEDOT:PSS, as it would behave in same manner as gold. In this regard, three recent works stating that PEDOT:PSS acts due to its electrical conductivity in the modulation of stem-cell differentiation should be interpreted conservatively [21
]. Moyen et al. hypothesized that the effect of PEDOT:PSS could be related to its specific stiffness [18
]. The coverage with collagen IV, which forms a relatively thick, 3D nonfibrillar network [49
], is not supportive of the idea that stiffness is behind the action of PEDOT:PSS. On the other hand, the hydrophilicity and specific surface chemistry of PEDOT:PSS could affect the conformation of adhered proteins, which in turn could modulate the interaction with living cells [49