ABSTRACT

Endogenous physical cue plays an enormous role in guiding the cellular response, apart from the biochemical interventions. That is the reason why in the field of regenerative medicine optimisation of the efficacy of physical cues always has been a matter of research. One of the physical cues that has attracted much attention of the researchers is the electrical stimulation, which is has been proven to have the potential of cellular differentiation and regeneration, along with other endogenous physical cues like shear [1, 2]. In this context, the efficacy of the electric field towards differentiation of stem cell along with a non-neuronal cell precursor appears to be crucial to establish the prospective. In the present study, it has been focused to understand the effectiveness of the electric field in combination with the influence of a customised extracellular environment towards regulating the neuronal behaviour and differentiation. This study reveals the promising effect in cellular differentiation of cocultured cells, together with application of customised extracellular biochemical cues on top of the electric field (Figure 1) [3].

A tailored lab-on-a-chip was designed with built-in electrodes to perform the planned set of experiments. The results were found to be reproducible, as the electrodes were fixed unlike the manually placed electrodes in case of conventional petridish cultures. In this study, it was identified that the neuronal differentiation of mouse precursor cells was enhanced when they were along with stem cells, under the influence of the electric field. The presence of surface adhesion of the cells with a customised surface-coating also found to enhance the efficacy of differentiation, with the electric field as it resembled similar physiological conditions of neural tissues. Results exhibit elevated expression of protein as well as the mRNA of the neuronal markers (Nestin, βIII-Tubulin, MAP2). Also the focal adhesion complex proteins (NCAM and vinculin), which is considered to maintain critical interaction between cell-to-surface, were found to have increased due to the electric field and biochemical stimulation. From this, the hypothesis could be established that these two physiological-survival cues are essential for differentiation and act synergistically onto the cellular genetic program. It is worthwhile to mention electric field alone failed to initiate the outcomes by manifesting a lower degree of differentiation (Figure 2). In corroboration of the outcomes, intracellular Ca²⁺ levels and inter cellular calcium oscillation were increased with the application of both of the cues (video file 1 and 2). These facts suggested that the intracellular signal to drive the cells towards neuronal differentiation was enhanced under the electric field stimulus which was potentiated more by the modified extracellular biochemical microenvironment. Addendum to these, adhesion of the cells to the surface coatings, which played the role of an intermediate signal transducer involving the downstream cascade reactions, were effective to guide non-neuronal lineages into neuronal path by overcoming the difficulty of neurogenic-differentiation.

Keywords: Lab-On-a-Chip, Electric field, Extracellular biochemical cues

Figure 1. Immunofluorescence image with actin(red)/ α-tubulin(green)/ nucleus(blue) staining show elongated cell according to the hypothesis and cells with neurite-like projections [3].

Figure 2. This specific marker expression has been assessed. (a) Cocultured cells exhibited neurite like projections with appreciable expression of βIII-tubulin, (b)Neural marker was absent in both electric field exposed and unexposed C2C12 monocultured cells. (c)Considerable amount of βIII-tubulin has been expressed for hMSc clusters, while the unexposed cells have failed to do so [3].

[1] Naskar S, Panda AK, Kumaran V, Mehta B, Basu B. Controlled Shear Flow Directs Osteogenesis on UHMWPE-Based Hybrid Nanobiocomposites in a Custom-Designed PMMA Microfluidic Device. ACS Applied Bio Materials. 2018;1:414-35.

[2] Naskar S, Kumaran V, Basu B. On The Origin of Shear Stress Induced Myogenesis Using PMMA Based Lab-on-Chip. ACS Biomaterials Science & Engineering. 2017.

[3] Naskar S, Kumaran V, Markandeya YS, Mehta B, Basu B. Neurogenesis-on-Chip: Electric field modulated transdifferentiation of human mesenchymal stem cell and mouse muscle precursor cell coculture. Biomaterials. 2020;226:119522.