Morphology and electrostatics play active role in neuronal differentiation processes on flexible conducting substrates

Abstract

This commentary discusses and summarizes the key highlights of our recently reported work entitled “Neuronal Differentiation of Embryonic Stem Cell Derived Neuronal Progenitors Can Be Regulated by Stretchable Conducting Polymers.” The prospect of controlling the mechanical-rigidity and the surface conductance properties offers a unique combination for tailoring the growth and differentiation of neuronal cells. We emphasize the utility of transparent elastomeric substrates with coatings of electrically conducting polymer to realize the desired substrate-characteristics for cellular development processes. Our study showed that neuronal differentiation from ES cells is highly influenced by the specific substrates on which they are growing. Thus, our results provide a better strategy for regulated neuronal differentiation by using such functional conducting surfaces.

Keywords: :

• neuronal cells
• conducting polymers
• stretched substrates
• electrostatics
• cell differentiation

This article refers to:

The ability to manipulate the vivid interactions between a cell and its underlying substrate has opened up interesting possibilities to tailor the physiological properties and behavior of the specific cell-type. Each of the processes defining the cellular systems development such as cell adhesion, proliferation and its differentiation have known to be affected by the environmental conditions.^1-4 The possibility to tamper the natural developmental machinery to direct the differentiation of stem cells toward a specific phenotype using various polymeric scaffolds has received much attention over the recent years.^5-10 Polymer surfaces offer a wide range of physical and mechanical attributes as well as microscopic features to enable growth, development and functioning of differentiating stem cells. Specifically, parameters such as adhesive strength, micro texture and nanotopography, surface wettability and stiffness can be controlled and engineered in the substrates.^4,11-13 Besides these features, it has also been realized that introduction of electronic conductivity can further provide a specific handle to control the differentiation or other physiological properties of the cell.^14,15

In vitro studies of the physiological properties of cells have shown that they can integrate and respond to a variety of biological, chemical and physical cues provided to them by the extra-cellular matrix.¹⁶ Cells bind to the ECM via focal adhesion complexes which are formed by the recruitment of many protein complexes and clustering of integrins.¹⁷ These focal adhesion complexes are linked to the actin networks which are the major components of the cytoskeletal framework of the cell. This structure provides the mechanical basis between the ECM and the cytoskeleton which allows the cells to exert traction forces that are transmitted to cell nucleus through intracellular pathways.¹⁷ Any alteration in the physical environment of the underlying matrix for example, a change in the ligand density or adhesivity alters the cell adhesion by modulating the focal adhesion complexes and this affects the cell spreading and migration.¹⁸ The topography of the substrate affects the adsorption of the surface proteins including fibronectin and collagen which help the cells to adhere to the substrate.^19-21 A preferential adsorption of proteins may take place since the topography of the substrate alters the surface wettability and the surface energy.²² The introduction of a conducting surface in form of a conducting polymer coating opens up additional vistas for directing cellular functions. The conducting polymer films offer a combination of conducting and a charged surface which can be utilized for preferential cell adhesion and directed growth.^23,24The unique chemical and ionic properties and the exceptional control over the morphology, surface chemistry, mechanical properties make polymer blends such as PEDOT:PSS as a possible choice for realization of biomedical devices.^25,26 The ionic properties of the conducting polymers can be easily modified by changing the doping concentration which provides a dynamic control over the release of biological molecules.^14,27 The introduction of direct electrostatic interactions originating from the charged regions and the conducting regions of appropriate length scales can have a profound effect. Neuronal cells have shown affinity for binding on conducting polymer surfaces and patterning of conducting regions have shown to be responsible for their directed growth.^28-30 Conducting polymers like PEDOT:PSS are biocompatible and have been functionalized with various peptides which gives exceptional control over cell attachment and neurite outgrowth.^31-33

The fabrication of different conducting polymer surfaces is generally done by directly dispensing commercially available dispersions available in aqueous and non-aqueous forms. The conductivity of conducting polymer films can also be further increased by introduction of suitable additives with appropriate drying and annealing conditions. Chemical and electrodeposition³⁴ methods of polymerization from monomers are other film forming routes. Chemical polymerization proceeds through either addition reactions, which are often stimulated by cations, anions or free radicals, or condensation reactions.¹⁴ This technique allows various modifications such as covalent attachments to the backbone. Substrate compatibility and post-fabrication thermal and miscellaneous processing issues are however some constraining factors.³⁵

The role of mechanical signaling and the biophysical interpretation of the ECM environment also plays a major role in directing stem cell differentiation to different lineages.^4,5,36 Although, stem cell differentiation could be controlled through chemical methods but purely physical methods which include altering substrate properties provide convenient routes to regenerative medicine.^11,12,37 Mesenchymal stem cell (MSC) lineage is influenced by the stiffness of substrate where the softer substrates induce neuronal growth while stiffer ones promote osetocyte lineage.^4,11 Differentiation of neuronal progenitor cells increases on softer substrates with young’s modulus similar to the native brain tissue.^12,38 Stem cell differentiation and self-renewal is also affected by the surface properties of the polymers as described previously.^39-41 Thus, the advent of changing the surface properties of polymers keeping the desired characteristics required for stem cell proliferation and differentiation is opening up interesting possibilities. The mechanical deformation of this media by stretching, bending, or twisting changes the cell-substrate interaction in a controlled manner. The changes in the cell-substrate interaction leads to a controlled change in the physiological functioning of the growing cells.

In a recent work from our laboratories, we demonstrated the effect of electroactivity and varying charge distribution produced by straining PEDOT:PSS (Poly[3,4-ethylenedioxythiophene] poly[styrenesulfonate]) coated SEBS (styrene ethylene butylene styrene) substrates on the differentiation of mouse D3 ES-NPs (embryonic stem cell derived neuronal progenitors).¹⁵ We also showed that the cellular distribution over the substrate is affected by the strain applied on the substrates. The work also emphasized on the reorganization of the actin fibers due to the electroactivity of the substrates and the strain applied on them.

These ES-NPs were differentiated on variety of surfaces including electroactive PEDOT:PSS coated SEBS substrates, SEBS substrates and glass coverslips which were used as controls in these studies.¹⁵ The formation of neurons from ES-NPs was confirmed by immunostaining with β-III tubulin which is a marker for immature neurons. It was seen that the neurons differentiated from ES-NPs on all the substrates but the differentiation potential of ES-NPs varied with the strain and electroactivity of the substrates. A key aspect in this work was that these studies were also performed on substrates systematically as they were stretched (and strained). The element of straining basically is a method to make the surface stiffer. The differentiation of ES-NPs was higher on strained SEBS substrates as compared with unstrained ones. An interesting set of observation in this study was that the differentiation of ES-NPs into neurons dramatically reduced on strained PEDOT:PSS substrates. The neurite length was also quantified and it supported the trend echoed by the differentiation potential of the ES-NPs. Neurons bore shorter neurites on highly strained conducting substrates while their length was much longer on the non-conducting SEBS substrates.¹⁵

It was noted that upon straining, besides the macroscopic quantities of stiffness and conductivity, the local morphology of the polymers also gets modified.⁴² This tended to have an effect on the differentiation and proliferation of ES-NPs. The thin sheets of neurons encompassed the non-conducting substrates but in case of conducting substrates the neurons tended to form clusters or aggregates on the conducting regions. This aggregate forming tendency increased with the strain on these electroactive substrates. The detailed analysis of the aggregates on these conducting substrates showed that the neurons were well-differentiated and formed inter-connecting networks at the interface with the substrates but the microstructure of the aggregates away from the substrate differed considerably.

We also introduce the concept of “defect patterning” which essentially implies the formation of mild defects/crack patterns upon straining which appear to act as nucleating sites in the growth process.¹⁵ The studies revealed that such defects develop on the PEDOT:PSS coated SEBS substrates over a strain regime, which does not affect the electrical continuity of the surface. These defects provide a suitable site along which the aggregates tend to spread. This trend is coherent with the analysis which indicates that these aggregates align along the defects on the strained conducting substrates. Thus, the cells are able to sense these cues on the surface of the substrates which may provide a convenient method for directional guidance of the growing neurons.

The morphology and surface conductance of the electroactive PEDOT:PSS coated SEBS substrates was varied by controlled straining of these substrates in the study. The application of strain leads to a change in the morphology of PEDOT domains to a more regular elliptical structures as compared with highly irregular ones. The controlled straining of the substrates also leads to a non-uniform distribution of these domains in the anionic PSS matrix (Fig. 1A and B).

Figure 1. Differentiation of ES-NPs on PEDOT:PSS/SEBS substrates (typical substrate shown on top-center [A]). The differentiation of neuronal progenitors and spreading of the differentiated neurons is strongly governed by the arrangement of conducting PEDOT domains in the PSS matrix which is controlled by uniaxial strain on these substrates as shown in the schematic (B) PEDOT domains interspersed in PSS matrix (C) Stretching of PEDOT:PSS/SEBS substrates leads to the elongation of PEDOT domains. These dispersed domains assume elliptical shape in the direction of stretching. (D) Differentiation of ES-NPs on PEDOT:PSS/SEBS substrates leads to evenly dispersed cells with many small aggregates. (E) Differentiation of ES-NPs on stretched PEDOT:PSS/SEBS substrates leads to the formation of larger cell aggregates and reduced cell spreading.

The spatial variability in the distribution and morphology of PEDOT domains was speculated as a major factor promoting the aggregation of neurons. The initial event of protein adsorption was limited to the area surrounding the PEDOT domains, and thus the cell adhesion was favored only on these areas of the substrates. Neuronal spreading was limited on these strained conducting substrates and the defects in the conducting layer provided the guided path for the aggregate spreading (Fig. 1C and D). The variability in the protein layer, necessary for cell adhesion, also affected the actin arrangement in the cells. Our studies also showed that the actin fibers were irregularly arranged in the cells present on the conducting substrates while the ones on the non-conducting substrates were well arranged. The arrangement of the actin fiber is important for maintenance of cell shape, regulation of focal adhesion complexes and generation of stable traction forces.^43,44 The change in either characteristic has been known to lead to the changes in the gene expression which control the differentiation of the progenitor cells.^4,11,34

Thus, the results indicate that the change in the distribution of the PEDOT domains might affect the adsorption of proteins necessary for cell adhesion. The confinement of the differentiating cells in the certain region leads to a decrease in the differentiation potential of ES-NPs and thus they produce much shorter neurites. The absence of these domains in the non-conducting substrates provides a uniform distribution of the adsorbed protein which lead to better spreading of the differentiating neurons.

Therefore, this study provides a useful method for controlling the neuronal differentiation of ES-NPs which is useful both from developmental biology perspective as well as in terms of biomedical applications. For instance, since the conducting polymer surface assists the progenitors to differentiate and proliferate in a manner of 3D rosette-like structures, this could be a convenient method for growing progenitors into neurons which can be readily used for clinical applications. But, further investigations are needed to understand the possible molecular pathways which take part in mechanotransduction events during the differentiation of the cells. A better understanding of the variation in the protein distribution with the change in the surface potential of the electroactive substrates is also needed since it would provide a much easier and viable route of controlled cell adhesion and proliferation. The understanding of the interaction of the neuronal progenitors with the electroactive and conducting ECM would definitely pave way toward the development of the next-generation of biomedical devices which are flexible and provide intimate contact between the cells and the underlying substrates.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

10.4161/org.27213