Bioelectric Potential in Next-Generation Organoids: Electrical Stimulation to Enhance 3D Structures of the Central Nervous System

To understand the relevance of these electrochemical conditions caused by exogenous electrical stimulation, we need a more detailed understanding of endogenous EFs in the human CNS. In vivo, EFs are present at the cellular level, at the level of the whole epithelium or tissue, and even the whole-organism or embryo, mediating development or regeneration (Levin et al., 2017). Mapping of bioelectric networks at each level is therefore a highly complex task. Studies of developmental bioelectricity are working to unveil these networks in small model organisms (Levin, 2014; Levin et al., 2017; Levin, 2019; Levin, 2021). Molecular mechanisms of EF-induced effects have been revealed by studying the proteome and transcriptome, as genomic regulation is powerfully connected to the bioelectric state. Through the expression of ion channels and regulators, bioelectric state can be genetically programmed. Correspondingly, through the downstream activation of transcription factors, bioelectric states may drive gene expression. However, the same bioelectric state of a cell may be achieved using various mechanisms. A multitude of ionic combinations or changes in transporter permeability may confer the same overall net effect on cellular charge and TEP. Therefore, multi-omics alone will not capture the complexity of bioelectric networks, and this data will require integration with both in silico computational modelling, and physiological measurements of EFs.

Once we better understand these studies we can begin to ask: to what extent are we recapitulating these endogenous electrical fields in vitro in our PSC-derived organoids?

Endogenous electric fields, variations in TEP and exogenous application of electrical stimulation have been linked to multiple canonical signalling networks and metabolic pathways (Figure 5C). In particular, EFs have been identified to regulate pathways involving phosphatase and kinase activity. Numerous reports have confirmed the ability of EFs to induce activation of Akt, and signal transduction of the PI3K pathway (Arocena et al., 2010; Meng et al., 2011; Wang et al., 2013; Dong et al., 2019; Yang et al., 2019; Wei et al., 2020). Additionally, multiple studies have identified various electrical field parameters to increase production of nitric oxide (NO) both in vivo and in vitro (Petrofsky et al., 2007; Kim et al., 2013; Trivedi et al., 2013; Wei et al., 2020). Specifically, EFs induce activating phosphorylation of eNOS, and levels of phosphorylation are proportional to electric field potential (Wei et al., 2020). PI3K inhibitors prevent EF-induced eNOS and Akt activation (Wei et al., 2020). In neuronal cells, PI3K/Akt signalling mediates EF-induced differentiation, dependent on expression of Achaete-scute homolog 1 (Ascl1) (Dong et al., 2019). In spinal cord injured rats, electrical stimulation was identified to promote neural survival through RhoA signalling and ERK1/2-Bcl-2 pathway (Joo et al., 2018). Phenotypically, increased RhoA signalling and ERK1 signalling results in increased neurite outgrowth and decreased apoptosis in the spinal cord of electrically stimulated rats (Joo et al., 2018). Other studies implicate Wnt signalling as a target for EF modulation, for example by promoting the migration and neurogenesis in EF-modulated rat brains through Wnt/GSK3β (Liu et al., 2013). Genetic manipulation of ion channels also determined EF-induced neuronal maturation in vivo to involve inhibition of canonical Wnt/beta-catenin signalling (Vitali et al., 2018).

However, another study found Wnt signalling alone was not sufficient to induce EF-specific effects on neural progenitor proliferation (Sefton et al., 2020).

Other, more transient signalling molecules may also function as downstream bioelectric signals. For example, the upregulation of calcium signalling has also been directly implicated in EF-induced neural fate determination (Yamada et al., 2007; Shin et al., 2017; Enayati et al., 2020). Reactive oxygen species (ROS), such as hydrogen peroxide (H2O2) are ubiquitous signalling molecules with defined roles in axial patterning, CNS development, differentiation and regeneration (Reviewed in Coffman and Su, 2019). NADPH oxidase, which catalyses H2O2 production, can be activated by exogenous electrical currents (Chatterjee et al., 2012; Li et al., 2013b). During amphibian regeneration, H2O2 and Vmem depolarisation overlap spatiotemporally. Studies using NADPH oxidase inhibitors during Xenopus regeneration suggest a two-way regulation mechanism intertwining redox reactions and electrical fields (Ferreira et al., 2016). The authors postulate that a change in membrane potential provides a rapid and dynamic signal for NADPH activation. Spatiotemporal H2O2 gradients exist throughout development and may therefore provide a blueprint for bioelectrical activity. Primary embryo-derived NSCs exhibit higher levels of mitochondrial and cytoplasmic ROS than committed NPCs (Khacho et al., 2016). This is mediated by the changing mitochondrial dynamics as NSCs commit to progenitor stage. Thus, increased ROS-mediated signalling functions to supress self-renewal and promote differentiation via Nuclear factor-erythroid factor 2-related factor 2 (Nrf2) signalling (Khacho et al., 2016). Accordingly, endogenous ROS signalling is important for the reprogramming of somatic cells to iPSCs, and ROS levels are stringently controlled during growth of iPSCs and ESCs in vitro, along with NSCs (Lee et al., 2012; Yang et al., 2013; Yeo et al., 2013).

As such, any effect of exogenous electrical fields on ROS production in PSC-derived cultures should be monitored, as stage-specific control of ROS production may be desirable. Build-up of by-products in PSC and organoid cultures should also be mitigated with immediate and frequent changing of culture medium after electrical stimulation.

A detailed review of bioelectric-driven molecular mechanisms governing development has recently been produced (George and Bates, 2022), and is beyond the scope of this review. However, the ability of EFs to influence canonical signal transduction and metabolic programming therefore provides a wealth of mechanisms by which electrical stimulation may enhance cell survival, migration, proliferation and differentiation of PSC-derived organoid cultures (Figure 5). Moreover, PSC-derived organoids provide micro-physiological systems that are amenable to EF manipulation in vitro, to aid delineation of molecular mechanisms directing EF-induced phenotypes. Downstream multi-omics analysis, including transcriptomics, proteomics and metabolomics, combined with functional analysis, such as electrophysiology will be key in identifying targets of bioelectricity and correlating this with phenotypic response. Advantageously, some EF-induced signals, such as ROS and Ca2+ may be rapidly and dynamically generated in response to electrical stimulation, allowing stage-specific control over PSC differentiation and organoid development.

Disease modelling and development of novel therapeutics are a major utility of CNS organoids. As a standalone therapy, electrical stimulation of the CNS is gaining increasing attention. It is therefore noteworthy to consider the applicability of organoids for disease modelling and therapeutics. Recently, ground-breaking studies demonstrated that tissue of brain and spinal cord tumours, gliomas, are electrically active and integrated into host neural tissue networks (Venkataramani et al., 2019; Venkatesh et al., 2019). This paved the way for electro-therapies to target gliomas with desirable effects on plasticity and neuromodulation, similar to that observed in brain stimulation for psychiatric and neurological disease (Reviewed in Sprugnoli et al., 2021). Previously, studies found primary-derived glioblastoma cells respond to EF modulation with migration, in a manner highly dependent on the surrounding 2D or 3D microenvironment (Huang et al., 2016). Glioblastoma organoids are producible from genetically-engineered cerebral organoids, and provide an ideal platform for exploration of this phenomena (Bian et al., 2018; Ogawa et al., 2018). However, studies utilising organoids to understand bioelectric-related disease phenotypes are very limited. Using iPSCs derived from schizophrenia patients, schizophrenia-cerebral organoids show an impaired response to electrical stimulation and ion channel-mediated depolarisation when compared to healthy controls (Kathuria et al., 2020). Given the use of deep-brain stimulation to treat schizophrenia and other psychiatric disorders, it would be informative to undertake longitudinal studies examining the response of organoid models of psychiatric disease to electrical stimulation in long-term culture.

Moreover, longer-term study of electrical activity may provide deeper insights into the complex mechanism of disrupted bioelectric networks in psychiatric disease, particularly subtle disturbances in EFs which may arise earlier in neurodevelopment, without the need for patient involvement. Nguyen et al. (2018) utilised iPSC-derived human 3D neural progenitor cells to study the effects of stimulation on Rett syndrome. As a rare but debilitating neurodevelopmental disorder, in vivo electrical stimulation has not been clinically applied for Rett syndrome patients. However, via culturing on conductive graphene scaffolds, the authors demonstrate electrical stimulation of diseased NPCs reduced disease phenotype (Nguyen et al., 2018). In concordance with previous studies, electrical stimulation promoted neurogenesis and neural maturation in Rett disease models, as in wildtype (Nguyen et al., 2018). As molecular responses to EF-modulation are further unveiled, the correlation of these to disease phenotypes may reveal bio-electric related pathologies and uncover new targets for electro-therapy.

If we are aiming to truly recapitulate development in the dish, then mapping the bioelectric networks in the body, and in our current organoid cultures in vitro is important. Whilst the lack of knowledge of mammalian bioelectricity networks is currently a limiting factor, it does not diminish the utility of EF modulation to enhance organoid development.

In the clinic, the application of EF to the CNS is beneficial (Balossier et al., 2015; Fu et al., 2015; Tao et al., 2016; Marquez-Chin and Popovic, 2020; Krauss et al., 2021; Sui et al., 2021; Karamian et al., 2022). To date, studies of EF modulation in vitro hold great promise for increasing the development and functionality of CNS derivative cells. However, they have focused on primary brain-derived cells rather PSC-derived organoids. PSC-derived organoids represent state-of-the-art systems for in vitro modelling of CNS organogenesis and disease. EF modulation of PSCs has demonstrated increases in neurogenic differentiation. Next, these findings should be translated into PSC-derived organoid cultures.

Whilst researchers are looking towards implementing advanced technologies to improve organoid differentiations, a simple electrical stimulation culture system could provide an additional, or missing, physiological cue. This could have important implications for increasing the utility of organoids in studies of development and disease, or applications in regenerative medicine and tissue engineering. As a cue for organoid differentiation, electrical stimulation is ready to be implemented in cell biology laboratories. Everything considered, bioelectricity, and the exploration of bioelectric phenomena both in vivo and in vitro is an exciting area to watch. As a greater understanding of bioelectric phenomena in vivo evolves, as will the utility of electrical stimulation as a tool in organoid generation. In parallel, PSC-derived CNS organoids provide the best in vitro model to unravel endogenous EFs in the human CNS. Based on the current body of research, we postulate exogenous application of EFs to brain organoids may increase cell proliferation, survival or differentiation of desired cell subtypes, whilst reducing death and necrosis. The optimisation of suitable stimulation parameters and field strength will be key in utilising EF manipulation for engineered brain tissue. Ideally, precise or stage-dependent application of electrical stimulation, or modulation of EFs by other means, could be implemented at key developmental timepoints to control organoid growth and maturation.