Bioelectric Signaling Research Articles

Electrode Droplet Microarray (eDMA): An Impedance Platform for Label-Free Parallel Monitoring of Cellular Drug Response in Nanoliter Droplets.

Label-free real-time monitoring of cellular behavior using impedance spectroscopy is important for drug development and toxicological assessments. Parallelization and miniaturization of such experiments are essential for increasing throughput and enabling experiments with low abundant stem or primary cells. Traditional methods are not miniaturized and require large volumes of reagents and number of cells, limiting their suitability for cost effective high-throughput screening of cells of limited availability. Here, the fabrication, optimization, and application of a bioelectrical signaling monitoring system - electrode droplet microarray (eDMA) are demonstrated. The eDMA platform is based on preparation of a hydrophilic-superhydrophobic patterns covering an array of individually addressable microelectrodes, which confines cells to individual microelectrodes, allowing for parallel, real-time, and label-free detection of cellular responses to drug treatments in nanoliter droplets. The real-time monitoring of cytotoxic effect of an anticancer drug is demonstrated over 48 h with real-time calculation of the half-inhibitory concentration (IC50) values through impedance spectroscopy. This demonstrates eDMA's ability to dynamically assess responses to various drugs in parallel at any given time point, which is crucial for functional personalized oncology. Specifically, the platform can be employed for monitoring anticancer drug toxicity using limited patient samples, where the miniaturization provided by eDMA is essential.

Identification of Distinct, Quantitative Pattern Classes from Emergent Tissue-Scale hiPSC Bioelectric Properties.

Bioelectric signals possess the ability to robustly control and manipulate patterning during embryogenesis and tissue-level regeneration. Endogenous local and global electric fields function as a spatial 'pre-pattern', controlling cell fates and tissue-scale anatomical boundaries; however, the mechanisms facilitating these robust multiscale outcomes are poorly characterized. Computational modeling addresses the need to predict in vitro patterning behavior and further elucidate the roles of cellular bioelectric signaling components in patterning outcomes. Here, we modified a previously designed image pattern recognition algorithm to distinguish unique spatial features of simulated non-excitable bioelectric patterns under distinct cell culture conditions. This algorithm was applied to comparisons between simulated patterns and experimental microscopy images of membrane potential (Vmem) across cultured human iPSC colonies. Furthermore, we extended the prediction to a novel co-culture condition in which cell sub-populations possessing different ionic fluxes were simulated; the defining spatial features were recapitulated in vitro with genetically modified colonies. These results collectively inform strategies for modeling multiscale spatial characteristics that emerge in multicellular systems, characterizing the molecular contributions to heterogeneity of membrane potential in non-excitable cells, and enabling downstream engineered bioelectrical tissue design.

Gap-junction-mediated bioelectric signaling required for slow muscle development and function in zebrafish

Bioelectric signaling, intercellular communication facilitated by membrane potential and electrochemical coupling, is emerging as a key regulator of animal development. Gap junction (GJ) channels can mediate bioelectric signaling by creating a fast, direct pathway between cells for the movement of ions and other small molecules. In vertebrates, GJ channels are formed by a highly conserved transmembrane protein family called the connexins. The connexin gene family is large and complex, creating challenges in identifying specific connexins that create channels within developing and mature tissues. Using the embryonic zebrafish neuromuscular system as a model, we identify a connexin conserved across vertebrate lineages, gjd4, which encodes the Cx46.8 protein, that mediates bioelectric signaling required for slow muscle development and function. Through mutant analysis and invivo imaging, we show that gjd4/Cx46.8 creates GJ channels specifically in developing slow muscle cells. Using genetics, pharmacology, and calcium imaging, we find that spinal-cord-generated neural activity is transmitted to developing slow muscle cells, and synchronized activity spreads via gjd4/Cx46.8 GJ channels. Finally, we show that bioelectrical signal propagation within the developing neuromuscular system is required for appropriate myofiber organization and that disruption leads to defects in behavior. Our work reveals a molecular basis for GJ communication among developing muscle cells and reveals how perturbations to bioelectric signaling in the neuromuscular system may contribute to developmental myopathies. Moreover, this work underscores a critical motif of signal propagation between organ systems and highlights the pivotal role of GJ communication in coordinating bioelectric signaling during development.

Conductive Microgel Annealed Scaffolds Enhance Myogenic Potential of Myoblastic Cells.

Conductive biomaterials may capture native or exogenous bioelectric signaling, but incorporation of conductive moieties is limited by cytotoxicity, poor injectability, or insufficient stimulation. Microgel annealed scaffolds are promising as hydrogel-based materials due to their inherent void space that facilitates cell migration and proliferation better than nanoporous bulk hydrogels. Conductive microgels aregenerated from poly(ethylene) glycol (PEG and poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT: PSS) to explore the interplay of void volume and conductivity on myogenic differentiation. PEDOT: PSS increasesmicrogel conductivity two-fold while maintaining stiffness, annealing strength, and viability of associated myoblastic cells. C2C12 myoblasts exhibitincreases in the late-stage differentiation marker myosin heavy chain as a function of both porosity and conductivity. Myogenin, an earlier marker, isinfluenced only by porosity. Human skeletal muscle-derived cells exhibitincreased Myod1, insulin like growth factor-1, and insulin-like growth factor binding protein 2 at earlier time points on conductive microgel scaffolds compared to non-conductive scaffolds. They also secretemore vascular endothelial growth factor at early time points and expressfactors that led to macrophage polarization patterns observe during muscle repair. These data indicate that conductivity aids myogenic differentiation of myogenic cell lines and primary cells, motivating the need for future translational studies to promote muscle repair.

Bioelectric networks: the cognitive glue enabling evolutionary scaling from physiology to mind.

Each of us made the remarkable journey from mere matter to mind: starting life as a quiescent oocyte ("just chemistry and physics"), and slowly, gradually, becoming an adult human with complex metacognitive processes, hopes, and dreams. In addition, even though we feel ourselves to be a unified, single Self, distinct from the emergent dynamics of termite mounds and other swarms, the reality is that all intelligence is collective intelligence: each of us consists of a huge number of cells working together to generate a coherent cognitive being with goals, preferences, and memories that belong to the whole and not to its parts. Basal cognition is the quest to understand how Mind scales-how large numbers of competent subunits can work together to become intelligences that expand the scale of their possible goals. Crucially, the remarkable trick of turning homeostatic, cell-level physiological competencies into large-scale behavioral intelligences is not limited to the electrical dynamics of the brain. Evolution was using bioelectric signaling long before neurons and muscles appeared, to solve the problem of creating and repairing complex bodies. In this Perspective, I review the deep symmetry between the intelligence of developmental morphogenesis and that of classical behavior. I describe the highly conserved mechanisms that enable the collective intelligence of cells to implement regulative embryogenesis, regeneration, and cancer suppression. I sketch the story of an evolutionary pivot that repurposed the algorithms and cellular machinery that enable navigation of morphospace into the behavioral navigation of the 3D world which we so readily recognize as intelligence. Understanding the bioelectric dynamics that underlie construction of complex bodies and brains provides an essential path to understanding the natural evolution, and bioengineered design, of diverse intelligences within and beyond the phylogenetic history of Earth.

Zebrafish Embryos Display Characteristic Bioelectric Signals during Early Development.

Bioelectricity is defined as endogenous electrical signaling mediated by the dynamic distribution of charged molecules. Recently, increasing evidence has revealed that cellular bioelectric signaling is critical for regulating embryonic development, regeneration, and congenital diseases. However, systematic real-time in vivo dynamic electrical activity monitoring of whole organisms has been limited, mainly due to the lack of a suitable model system and voltage measurement tools for in vivo biology. Here, we addressed this gap by utilizing a genetically stable zebrafish line, Tg (ubiquitin: ASAP1), and ASAP1 (Accelerated sensor of action potentials 1), a genetically encoded voltage indicator (GEVI). With light-sheet microscopy, we systematically investigated cell membrane potential (Vm) signals during different embryonic stages. We found cells of zebrafish embryos showed local membrane hyperpolarization at the cleavage furrows during the cleavage period of embryogenesis. This signal appeared before cytokinesis and fluctuated as it progressed. In contrast, whole-cell transient hyperpolarization was observed during the blastula and gastrula stages. These signals were generally limited to the superficial blastomere, but they could be detected within the deeper cells during the gastrulation period. Moreover, the zebrafish embryos exhibit tissue-level cell Vm signals during the segmentation period. Middle-aged somites had strong and dynamic Vm fluctuations starting at about the 12-somite stage. These embryonic stage-specific characteristic cellular bioelectric signals suggest that they might play a diverse role in zebrafish embryogenesis that could underlie human congenital diseases.

Bioelectric Signaling: Role of Bioelectricity in Directional Cell Migration in Wound Healing.

In wound healing, individual cells' behaviors coordinate movement toward the wound center to restore small or large barrier defects. The migration of epithelial cells as a continuous sheet structure is one of the most important processes by which the skin barrier is restored. How such multicellular and tissue level movement is initiated upon injury, coordinated during healing, and stopped when wounds healed has been a research focus for decades. When skin is wounded, the compromised epithelial barrier generates endogenous electric fields (EFs), produced by ion channels and maintained by cell junctions. These EFs are present across wounds, with the cathodal pole at the wound center. Epithelial cells detect minute EFs and migrate directionally in response to electrical signals. It has long been postulated that the naturally occurring EFs facilitate wound healing by guiding cell migration. It is not until recently that experimental evidence has shown that large epithelial sheets of keratinocytes or corneal epithelial cells respond to applied EFs by collective directional migration. Although some of the mechanisms of the collective cell migration are similar to those used by isolated cells, there are unique mechanisms that govern the coordinated movement of the cohesive sheet. We will review the understanding of wound EFs and how epithelial cells and other cells important to wound healing respond to the electric signals individually as well as collectively. Mounting evidence suggests that wound bioelectrical signaling is an important mechanism in healing. Critical understanding and proper exploitation of this mechanism will be important for better wound healing and regeneration.

Recent Development of Conductive Hydrogels for Tissue Engineering: Review and Perspective.

In recent years, tissue engineering techniques have been rapidly developed and offer a new therapeutic approach to organ or tissue damage repair. However, most of tissue engineering scaffolds are nonconductive and cannot establish effective electrical coupling with tissue for the electroactive tissues. Electroconductive hydrogels (ECHs) have received increasing attention in tissue engineering owing to their electroconductivity, biocompatibility, and high water content. In vitro, ECHs can not only promote the communication of electrical signals between cells, but also mediate the adhesion, proliferation, migration, and differentiation of different kinds of cells. In vivo, ECHs can transmit the electric signal to electroactive tissues and activate bioelectrical signaling pathways to promote tissue repair. As a result, implanting ECHs into damaged tissues can effectively reconstruct physiological functions related to electrical conduction. In this review, an overview about the classifications and the fabrication methods of ECHs is first presented. And then, the applications of ECHs in tissue engineering, including cardiac, nerve, skin, and skeletal muscle tissue, are highlighted. At last, some rational guidelines for designing ECHs toward clinical applications are provided.

Call for Special Issue Papers: Bioelectrical Signaling via Gap Junctions: Deadline for Manuscript Submission: March 1, 2023.

Call for Special Issue Papers: Bioelectrical Signaling via Gap Junctions: Deadline for Manuscript Submission: March 1, 2023.

Unveiling the morphogenetic code: A new path at the intersection of physical energies and chemical signaling.

In this editorial, we discuss the remarkable role of physical energies in the control of cell signaling networks and in the specification of the architectural plan of both somatic and stem cells. In particular, we focus on the biological relevance of bioelectricity in the pattern control that orchestrates both developmental and regenerative pathways. To this end, the narrative starts from the dawn of the first studies on animal electricity, reconsidering the pioneer work of Harold Saxton Burr in the light of the current achievements. We finally discuss the most recent evidence showing that bioelectric signaling is an essential component of the informational processes that control pattern specification during embryogenesis, regeneration, or even malignant transformation. We conclude that there is now mounting evidence for the existence of a Morphogenetic Code, and that deciphering this code may lead to unprecedented opportunities for the development of novel paradigms of cure in regenerative and precision medicine.

Bioelectric signaling and the control of cardiac cell identity in response to mechanical forces.

Developing cardiovascular systems use mechanical forces to take shape, but how ubiquitous blood flow forces instruct local cardiac cell identity is still unclear. By manipulating mechanical forces in vivo, we show here that shear stress is necessary and sufficient to promote valvulogenesis. We found that valve formation is associated with the activation of an extracellular adenosine triphosphate (ATP)–dependent purinergic receptor pathway, specifically triggering calcium ion (Ca2+) pulses and nuclear factor of activated T cells 1 (Nfatc1) activation. Thus, mechanical forces are converted into discrete bioelectric signals by an ATP-Ca2+-Nfatc1–mechanosensitive pathway to generate positional information and control valve formation.

Beyond Neurons: Long Distance Communication in Development and Cancer

Cellular communication is important in all aspects of tissue and organism functioning, from the level of single cells, two discreet populations, and distant tissues of the body. Long distance communication networks integrate individual cells into tissues to maintain a complex organism during development, but when communication between cells goes awry, disease states such as cancer emerge. Herein we discuss the growing body of evidence suggesting that communication methods known to be employed by neurons, also exist in other cell types. We identify three major areas of long-distance communication: bioelectric signaling, tunneling nanotubes (TNTs), and macrophage modulation of networks, and draw comparisons about how these systems operate in the context of development and cancer. Bioelectric signaling occurs between cells through exchange of ions and tissue-level electric fields, leading to changes in biochemical gradients and molecular signaling pathways to control normal development and tumor growth and invasion in cancer. TNTs transport key morphogens and other cargo long distances, mediating electrical coupling, tissue patterning, and malignancy of cancer cells. Lastly macrophages maintain long distance signaling networks through trafficking of vesicles during development, providing communication relays and priming favorable microenvironments for cancer metastasis. By drawing comparisons between non-neural long distance signaling in the context of development and cancer we aim to encourage crosstalk between the two fields to cultivate new hypotheses and potential therapeutic strategies.

Bringing Microbiology to Light: Toward All-Optical Electrophysiology in Bacteria.

The observation of neuron-like behavior in bacteria, such as the occurrence of electric spiking and extended bioelectric signaling, points to the role of membrane dynamics in prokaryotes. Electrophysiology of bacteria, however, has been overlooked for long time, due to the difficulties in monitoring bacterial bioelectric phenomena with those probing techniques that are commonly used for eukaryotes. Optical technologies can allow a paradigm shift in the field of electrophysiology of bacteria, as they would permit to elicit and monitor signaling rapidly, remotely, and with high spatiotemporal precision. In this perspective, we discuss about the potentiality of light interrogation methods in microbiology, encouraging the development of all-optical electrophysiology of bacteria.

Bioelectric signaling as a unique regulator of development and regeneration.

It is well known that electrical signals are deeply associated with living entities. Much of our understanding of excitable tissues is derived from studies of specialized cells of neurons or myocytes. However, electric potential is present in all cell types and results from the differential partitioning of ions across membranes. This electrical potential correlates with cell behavior and tissue organization. In recent years, there has been exciting, and broadly unexpected, evidence linking the regulation of development to bioelectric signals. However, experimental modulation of electrical potential can have multifaceted and pleiotropic effects, which makes dissecting the role of electrical signals in development difficult. Here, I review evidence that bioelectric cues play defined instructional roles in orchestrating development and regeneration, and further outline key areas in which to refine our understanding of this signaling mechanism.

Bioelectrical approaches to cancer as a problem of the scaling of the cellular self

One lens with which to understand the complex phenomenon of cancer is that of developmental biology. Cancer is the inevitable consequence of a breakdown of the communication that enables individual cells to join into computational networks that work towards large-scale, morphogenetic goals instead of more primitive, unicellular objectives. This perspective suggests that cancer may be a physiological disorder, not necessarily due to problems with the genetically-specified protein hardware. One aspect of morphogenetic coordination is bioelectric signaling, and indeed an abnormal bioelectric signature non-invasively reveals the site of incipient tumors in amphibian models. Functionally, a disruption of resting potential states triggers metastatic melanoma phenotypes in embryos with no genetic defects or carcinogen exposure. Conversely, optogenetic or molecular-biological modulation of bioelectric states can override powerful oncogenic mutations and prevent or normalize tumors. The bioelectrically-mediated information flows that harness cells toward body-level anatomical outcomes represent a very attractive and tractable endogenous control system, which is being targeted by emerging approaches to cancer.

The effects of membrane potential and extracellular matrix composition on vascular differentiation of cardiac progenitor cells

Historically, the field of tissue engineering has been adept at modulating the chemical and physical microenvironment. This approach has yielded significant progress, but it is imperative to further integrate our understanding of other fundamental cell signaling paradigms into tissue engineering methods. Bioelectric signaling has been demonstrated to be a vital part of tissue development, regeneration, and function across organ systems and the extracellular matrix is known to alter the bioelectric properties of cells. Thus, there is a need to bolster our understanding of how matrix and bioelectric signals interact to drive cell phenotype. We examine how cardiac progenitor cell differentiation is altered by simultaneous changes in both resting membrane potential and extracellular matrix composition. Pediatric c-kit+ cardiac progenitor cells were differentiated on fetal or adult cardiac extracellular matrix while being treated with drugs that alter resting membrane potential. Smooth muscle gene expression was increased with depolarization and decreased with hyperpolarization while endothelial and cardiac expression were unchanged. Early smooth muscle protein expression is modified by matrix developmental age, with fetal ECM appearing to amplify the effects of resting membrane potential. Thus, combining matrix composition and bioelectric signaling represents a potential alternative for guiding cell behavior in tissue engineering and regenerative medicine.

The synergistic effect of biomimetic electrical stimulation and extracellular-matrix-mimetic nanopattern for upregulating cell activities

Endogenous bioelectric signaling and the extracellular matrix (ECM) are factors that have a great effect on the performance of cellular functions. Presenting an experimental platform to confirm the synergy effects of an electrical stimulation, which simulates endogenous bioelectricity, and nanopatterns that can be precisely fabricated in various patterns sizes makes it possible to consider those factors effectively. Herein, we have performed a comparison of cellular response to each of general electrical stimulation and biomimetic electrical stimulation (BES) and demonstrated the synergy effects of electrical stimulation and ECM-mimetic nanopatterns. BES has provided the most remarkable proliferation among different types of electrical stimulation and upregulated the behavior of cells through synergy effects with ECM-mimetic nanopatterns. Thus, it is believed that using the synergy effects of BES and ECM-like nanopatterns has broad applications in the biomedical field, such as cell culture with electrical stimulation, induction of cell growth, tissue repair, etc.

SCHEEPDOG: Programming Electric Cues to Dynamically Herd Large-Scale Cell Migration.

Directed cell migration is critical across biological processes spanning healing to cancer invasion, yet no existing tools allow real-time interactive guidance over such migration. We present a new bioreactor that harnesses electrotaxis-directed cell migration along electric field gradients-by integrating four independent electrodes under computer control to dynamically program electric field patterns, and hence steer cell migration. Using this platform, we programmed and characterized multiple precise, two-dimensional collective migration maneuvers in renal epithelia and primary skin keratinocyte ensembles. First, we demonstrated on-demand, 90-degree collective turning. Next, we developed a universal electrical stimulation scheme capable of programming arbitrary 2D migration maneuvers such as precise angular turns and migration in a complete circle. Our stimulation scheme proves that cells effectively time-average electric field cues, helping to elucidate the transduction timescales in electrotaxis. Together, this work represents an enabling platform for controlling cell migration with broad utility across many cell types.

HCN2 Channel-Induced Rescue of Brain Teratogenesis via Local and Long-Range Bioelectric Repair.

Embryonic exposure to the teratogen nicotine results in brain defects, by disrupting endogenous spatial pre patterns necessary for normal brain size and patterning. Extending prior work in Xenopus laevis that showed that misexpression of ion channels can rescue morphogenesis, we demonstrate and characterize a novel aspect of developmental bioelectricity: channel-dependent repair signals propagate long-range across the embryo. We show that distal HCN2 channel misexpression and distal transplants of HCN2-expressing tissue, non-cell-autonomously reverse profound defects, rescuing brain anatomy, gene expression, and learning. Moreover, such rescue can be induced by small-molecule HCN2 channel activators, even with delayed treatment initiation. We present a simple, versatile computational model of bioelectrical signaling upstream of key patterning genes such as OTX2 and XBF1, which predicts long-range repair induced by ion channel activity, and experimentally validate the predictions of this model. Our results and quantitative model identify a powerful morphogenetic control mechanism that could be targeted by future regenerative medicine exploiting ion channel modulating drugs approved for human use.

A Meta-Analysis of Bioelectric Data in Cancer, Embryogenesis, and Regeneration.

Developmental bioelectricity is the study of the endogenous role of bioelectrical signaling in all cell types. Resting potentials and other aspects of ionic cell physiology are known to be important regulatory parameters in embryogenesis, regeneration, and cancer. However, relevant quantitative measurement and genetic phenotyping data are distributed throughout wide-ranging literature, hampering experimental design and hypothesis generation. Here, we analyze published studies on bioelectrics and transcriptomic and genomic/phenotypic databases to provide a novel synthesis of what is known in three important aspects of bioelectrics research. First, we provide a comprehensive list of channelopathies-ion channel and pump gene mutations-in a range of important model systems with developmental patterning phenotypes, illustrating the breadth of channel types, tissues, and phyla (including man) in which bioelectric signaling is a critical endogenous aspect of embryogenesis. Second, we perform a novel bioinformatic analysis of transcriptomic data during regeneration in diverse taxa that reveals an electrogenic protein to be the one common factor specifically expressed in regeneration blastemas across Kingdoms. Finally, we analyze data on distinct Vmem signatures in normal and cancer cells, revealing a specific bioelectrical signature corresponding to some types of malignancies. These analyses shed light on fundamental questions in developmental bioelectricity and suggest new avenues for research in this exciting field.