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Bioelectric Control of Stem Cell Functions
Abstract
The field of stem cell biology has gained momentum over the past decade
and half due to its potential to cure damaged and diseased tissues and
shed light on the processes by which a single fertilized egg gives rise to
the remarkable complexity of the embryonic body. Hopes for biomedical
applications are fueled by the detection of resident adult stem cell
populations in almost all tissues, including the central nervous system.
The majority of efforts to understand and control the cues that regulate
embryonic stem cell differentiation into various tissues have focused on
biochemical signals and transcriptional networks. However, bioelectric
signals mediated by slow changes in ion fl ows and transmembrane voltage
gradients encode patterning information in physiological networks that
guide embryonic morphogenesis, regeneration, and cancer suppression.
Recent molecular work has begun to unravel the mechanisms of endogenous
bioelectric infl uences on stem cell function. Here, we discuss fundamental
properties of bioelectric signals, the mechanisms by which they regulate
stem cell maintenance and functional differentiation, and the prospects of
manipulation of such signals for therapies in regenerative medicine.
... In addition to well-known genetic and biochemical signals, spatiotemporal changes in membrane voltage across somatic cells (bioelectric signals) regulate many aspects of large-scale embryonic patterning. [21][22][23][24][25][26][27][28] Channelopathies, 29 syndromes developing from mutations in ion channel genes, often cause brain and eye anomalies, strongly implicating bioelectricity as an important regulator of brain and eye development. Using voltage-sensitive dyes, 30 developing Xenopus embryos were shown to have dynamic spatiotemporal changes in the ectodermal membrane voltage now known as the ''electric face.'' ...
... The dishes were then moved to the light-tight box, either covered (control) or uncovered, or returned to the 18°C incubator to incubate in dark conditions (control). Xenopus embryos were exposed to 2% ethanol in three regimens associated with neural development: early OPTOGENETICS FOR FASD 261 (stage 9-23), late (stage [23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38][39][40], and full (stage 9-40). Brain and eye morphology of these embryos was assessed after they had developed to stage 45 ( Fig. 1). ...
... To discover whether the ChR2 D156A rescue of ethanolinduced brain and eye defects occurs in a local or nonlocal (noncell autonomous) manner, we targeted the mRNA to tadpole from embryos exposed to ethanol (2%-stage 9-40) and microinjected with channel rhodopsin (ChR2 D156A ) mRNA in both blastomeres at two-cell stage, showing severe brain morphology defects as indicated by magenta arrowheads. Cyan brackets indicate unaffected HB, (C) tadpole from embryos exposed to ethanol (2%-stage 9-40), microinjected with channel rhodopsin (ChR2 D156A ) mRNA in both blastomeres at two-cell stage, and exposed to blue light (stage 9-40), showing restoration of brain pattern with intact nostrils (blue arrowheads), distinct FB (orange bracket), MB (yellow bracket), and HB (cyan brackets), (D) tadpole from embryos exposed to ethanol (2%-stage 9-40), microinjected with channel rhodopsin (ChR2 D156A ) mRNA in both blastomeres at two-cell stage, and exposed to blue light for a shorter duration (stage 9-23 or stage [23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38][39][40], showing brain morphology defects as indicated by magenta arrowheads. Cyan brackets indicate unaffected HB. (E) Quantification of tadpoles with malformed brain phenotype in control (untreated and uninjected) embryos, embryos exposed to ethanol (2%-stage 9-40) with microinjection of channel rhodopsin (ChR2 D156A ) mRNA in both blastomeres at two-cell stage. ...
Background:
Embryonic exposure to the teratogen ethanol leads to dysmorphias, including eye and brain morphology defects associated with fetal alcohol spectrum disorder (FASD). Exposure of Xenopus laevis embryos to ethanol leads to similar developmental defects, including brain and eye dysmorphism, confirming our work and the work of others showing Xenopus as a useful system for studies of the brain and eye birth defects associated with FASD. Several targets of ethanol action have been hypothesized, one being regulation of Kir2.1 potassium channel. Endogenous ion fluxes and membrane voltage variation (bioelectric signals) have been shown to be powerful regulators of embryonic cell behaviors that are required for correct brain and eye morphology. Disruptions to these voltage patterns lead to spatially correlated disruptions in gene expression patterns and corresponding morphology.
Materials and Methods:
Here, we use controlled membrane voltage modulation to determine when and where voltage modulation is sufficient to rescue ethanol-induced brain and eye defects in Xenopus embryos.
Results:
We found (1) that modulating membrane voltage using light activation of the channelrhodopsin-2 variant D156A rescues ethanol exposed embryos, resulting in normal brain and eye morphologies; (2) hyperpolarization is required for the full duration of ethanol exposure; (3) hyperpolarization of only superficial ectoderm is sufficient for this effect; and(4) the rescue effect acts at a distance.
Conclusions:
These results, particularly the last, raise the exciting possibility of using bioelectric modulation to treat ethanol-induced brain and eye birth defects, possibly with extant ion channel drugs already prescribed to pregnant women. This may prove to be a simple and cost-effective strategy for reducing the impact of FASD.
... Regulation of resting potential (Vmem) of a wide variety of cell types plays an important role in embryogenesis, regenerative response, and cancer (Adams and Levin, 2013;Blackiston et al., 2009;Borgens et al., 1977b;Levin, 2013b, 2014;Levin, 2014a;Lobikin et al., 2012;McCaig et al., 2005;Pai and Levin, 2014;Pullar, 2011;Tseng and Levin, 2013b). In order to understand the role of bioelectricity in pattern formation, and to harness this signaling modality for biomedicine, it is important to understand the transcriptional networks downstream of specific Vmem change. ...
Endogenous bioelectric signaling via changes in cellular resting potential (Vmem) is a key regulator of patterning during regeneration and embryogenesis in numerous model systems. Depolarization of Vmem has been functionally implicated in de-differentiation, tumorigenesis, anatomical re-specification, and appendage regeneration. However, no unbiased analyses have been performed to understand genome-wide transcriptional responses to Vmem change in vivo. Moreover, it is unknown which genes or gene networks represent conserved targets of bioelectrical signaling across different patterning contexts and species. Here, we use microarray analysis to comparatively analyze transcriptional responses to Vmem depolarization. We compare the response of the transcriptome during embryogenesis (Xenopus development), regeneration (axolotl regeneration), and stem cell differentiation (human mesenchymal stem cells in culture) to identify common networks across model species that are associated with depolarization. Both sub-network enrichment and PANTHER analyses identified a number of key genetic modules as targets of Vmem change, and also revealed important (well-conserved) commonalities in bioelectric signal transduction, despite highly diverse experimental contexts and species. Depolarization regulates specific transcriptional networks across all three germ layers (ectoderm, mesoderm and endoderm) such as cell differentiation and apoptosis, and this information will be used for developing mechanistic models of bioelectric regulation of patterning. Moreover, our analysis reveals that Vmem change regulates transcripts related to important disease pathways such as cancer and neurodegeneration, which may represent novel targets for emerging electroceutical therapies.This article is protected by copyright. All rights reserved
... The authors suggest that this system may form a code, given that each cell has not one but many domains of Vmem along its surface, and so the spatial distribution of voltage values could form a rich combinatorial code. Vmem gradients form an important signal for modulating stem cell proliferation and differentiation (Pai and Levin, 2014). The authors conclude " . . . ...
In a series of recent papers, Levin and his coworkers (Levin, 2009, 2012; Adams and Levin, 2012; Tseng and Levin, 2012, 2013) introduced the concept of a morphogenetic code based on bioelectrical signaling between cells. They showed that patterns of resting potentials (Vmem) in non-excitable cells act as instructive signals during embryogenesis, regeneration and cancer suppression in a wide range of tissues. We will present the case that telocytes (TCs) may play an essential role in morphogenetic bioelectrical signaling.
... The authors suggest that this system may form a code, given that each cell has not one but many domains of Vmem along its surface, and so the spatial distribution of voltage values could form a rich combinatorial code. Vmem gradients form an important signal for modulating stem cell proliferation and differentiation (Pai and Levin, 2014). The authors conclude " . . . ...
In a series of recent papers, Levin and his coworkers (Levin, 2009, 2012; Adams and Levin, 2012; Tseng and Levin, 2012, 2013) introduced the concept of a morphogenetic code based on bioelectrical signaling between cells. They showed that patterns of resting potentials (Vmem) in non-excitable cells act as instructive signals during embryogenesis, regeneration and cancer suppression in a wide range of tissues. We will present the case that telocytes (TCs) may play an essential role in morphogenetic bioelectrical signaling.
All living cells maintain a charge distribution across their cell membrane (membrane potential) by carefully controlled ion fluxes. These bioelectric signals regulate cell behavior (such as migration, proliferation, differentiation) as well as higher-level tissue and organ patterning. Thus, voltage gradients represent an important parameter for diagnostics as well as a promising target for therapeutic interventions in birth defects, injury, and cancer. However, despite much progress in cell and molecular biology, little is known about bioelectric states in human stem cells. Here, we present simple methods to simultaneously track ion dynamics, membrane voltage, cell morphology, and cell activity (pH and ROS), using fluorescent reporter dyes in living human neurons derived from induced neural stem cells (hiNSC). We developed and tested functional protocols for manipulating ion fluxes, membrane potential, and cell activity, and tracking neural responses to injury and reinnervation in vitro. Finally, using morphology sensor, we tested and quantified the ability of physiological actuators (neurotransmitters and pH) to manipulate nerve repair and reinnervation. These methods are not specific to a particular cell type and should be broadly applicable to the study of bioelectrical controls across a wide range of combinations of models and endpoints.
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