visualizing-age-dependent-electrotaxis-human-adipose-derived-stem

Visualizing Age-Dependent Electrotaxis of Human Adipose-Derived Stem Cells Under Direct Current Electric Fields

Human adipose-derived stem cells (hADSCs) are pivotal for tissue regeneration and wound healing, and their directed migration is a prerequisite for ...

University of California, Davis

Weak electric fields guide cell migration, known as galvanotaxis/electrotaxis. The sensor(s) cells use to detect the fields remain elusive. Here we perform a large-scale screen using an RNAi library targeting ion transporters in human cells. We identify 18 genes that show either defective or increased galvanotaxis after knockdown. Knockdown of the KCNJ15 gene (encoding inwardly rectifying K(+) channel Kir4.2) specifically abolishes galvanotaxis, without affecting basal motility and directional migration in a monolayer scratch assay. Depletion of cytoplasmic polyamines, highly positively charged small molecules that regulate Kir4.2 function, completely inhibits galvanotaxis, whereas increase of intracellular polyamines enhances galvanotaxis in a Kir4.2-dependent manner. Expression of a polyamine-binding defective mutant of KCNJ15 significantly decreases galvanotaxis. Knockdown or inhibition of KCNJ15 prevents phosphatidylinositol 3,4,5-triphosphate (PIP3) from distributing to the leading edge. Taken together these data suggest a previously unknown two-molecule sensing mechanism in which KCNJ15/Kir4.2 couples with polyamines in sensing weak electric fields.

KCNJ15/Kir4.2 couples with polyamines to sense weak extracellular electric fields in galvanotaxis.

Cell fragments devoid of the nucleus and major organelles are found in physiology and pathology, for example platelets derived from megakaryocytes, and cell fragments from white blood cells and glioma cells. Platelets exhibit active chemotaxis. Fragments from white blood cells display chemotaxis, phagocytosis, and bactericidal functions. Signaling mechanisms underlying migration of cell fragments are poorly understood. Here we used fish keratocyte fragments and demonstrated striking differences in signal transduction in migration of cell fragments and parental cells in a weak electric field. cAMP or cGMP agonists completely abolished directional migration of fragments, but had no effect on parental cells. The inhibition effects were prevented by pre-incubating with cAMP and cGMP antagonists. Blocking cAMP and cGMP downstream signaling by inhibition of PKA and PKG also recovered fragment galvanotaxis. Both perturbations confirmed that the inhibitory effect was mediated by cAMP or cGMP signaling. Inhibition of cathode signaling with PI3K inhibitor LY294002 also prevented the effects of cAMP or cGMP agonists. Our results suggest that cAMP and cGMP are essential for galvanotaxis of cell fragments, in contrast to the signaling mechanisms in parental cells.

cAMP and cGMP Play an Essential Role in Galvanotaxis of Cell Fragments.

Recent studies have demonstrated distinctive motility and responses to extracellular cues of cells in isolation, cells collectively in groups, and cell fragments. Here we provide a protocol for generating cell sheets, isolated cells, and cell fragments of keratocytes from zebrafish scales. The protocol starts with a comprehensive fish preparation, followed by critical steps for scale processing and subsequent cell sheet generation, single cell isolation, and cell fragment induction, which can be accomplished in just 3 days including a 36-48 h incubation time. Compared to other approaches that usually produce single cells only or together with either fragments or cell groups, this facile and reliable methodology allows generation of all three motile forms simultaneously. With the powerful genetics in zebrafish our model system offers a useful tool for comparison of the mechanisms by which cell sheets, single cells, and cell fragments respond to extracellular stimuli.

An Experimental Model for Simultaneous Study of Migration of Cell Fragments, Single Cells, and Cell Sheets.

Stationary symmetrical fish keratocyte cells break symmetry and become motile spontaneously but slowly. We found that applying electric field (EF) accelerates the polarization by an order of magnitude. While spontaneously polarized cells move persistently for hours, the EF-induced polarity is lost in a majority of cells when the EF is switched off. However, if the EF is applied for a long time and then switched off, the majority of cell move stably. Myosin inhibition abolishes spontaneous polarization, but does not slow down EF-induced polarization, and after the EF is turned off, motility does not stop; however, the cell movements are erratic. Our results suggest that the EF rapidly polarizes the cells, but that resulting polarization becomes stable slowly, and that the EF bypasses the requirement for myosin action in motility initiation.

Electric fields accelerate cell polarization and bypass myosin action in motility initiation.

Published studies have reported conflicting and heterogeneous results regarding the association between human leukocyte antigen (HLA)-DRB1 polymorphisms and alopecia areata (AA). This study aimed to review and quantitatively analyze the association between HLA-DRB1 polymorphisms and AA.

HLA-DRB1 polymorphisms and alopecia areata disease risk: A systematic review and meta-analysis.

Proper control of cell migration is critically important in many biologic processes, such as wound healing, immune surveillance, and development. Much progress has been made in the initiation of cell migration; however, little is known about termination and sometimes directional reversal. During active cell migration, as in wound healing, development, and immune surveillance, the integrin expression profile undergoes drastic changes. Here, we uncovered the extensive regulatory and even opposing roles of integrins in directional cell migration in electric fields (EFs), a potentially important endogenous guidance mechanism. We established cell lines that stably express specific integrins and determined their responses to applied EFs with a high throughput screen. Expression of specific integrins drove cells to migrate to the cathode or to the anode or to lose migration direction. Cells expressing αMβ2, β1, α2, αIIbβ3, and α5 migrated to the cathode, whereas cells expressing β3, α6, and α9 migrated to the anode. Cells expressing α4, αV, and α6β4 lost directional electrotaxis. Manipulation of α9 molecules, one of the molecular directional switches, suggested that the intracellular domain is critical for the directional reversal. These data revealed an unreported role for integrins in controlling stop, go, and reversal activity of directional migration of mammalian cells in EFs, which might ensure that cells reach their final destination with well-controlled speed and direction.-Zhu, K., Takada, Y., Nakajima, K., Sun, Y., Jiang, J., Zhang, Y., Zeng, Q., Takada, Y., Zhao, M. Expression of integrins to control migration direction of electrotaxis.

Expression of integrins to control migration direction of electrotaxis.

Direct current (DC) electrical stimulation has been shown to have remarkable effects on regulating cell behaviors. Translation of this technology to clinical uses, however, has to overcome several obstacles, including Joule heat production, changes in pH and ion concentration, and electrode products that are detrimental to cells. Application of DC voltages in thick tissues where their thickness is >0.8 mm caused significant changes in temperature, pH, and ion concentrations. In this study, we developed a multifield and -chamber electrotaxis chip, and various stimulation schemes to determine effective and safe stimulation strategies to guide the migration of human vascular endothelial cells. The electrotaxis chip with a chamber thickness of 1 mm allows 10 voltages applied in one experiment. DC electric fields caused detrimental effects on cells in a 1 mm chamber that mimicking 3D tissue with a decrease in cell migration speed and an increase in necrosis and apoptosis. Using the chip, we were able to select optimal stimulation schemes that were effective in guiding cells with minimal detrimental effects. This experimental system can be used to determine optimal electrical stimulation schemes for cell migration, survival with minimal detrimental effects on cells, which will facilitate to bring electrical stimulation for  use.

Optimization of Electrical Stimulation for Safe and Effective Guidance of Human Cells.

Intracellular signaling dynamics play fundamental roles in cell biology. Precise modulation of the amplitude, duration, and frequency of signaling activation will be a powerful approach to investigate molecular mechanisms as well as to engineer signaling to control cell behaviors. Here, we showed a practical approach to achieve precise amplitude modulation (AM), frequency modulation (FM), and duration modulation (DM) of MAP kinase activation. Alternating current (AC) electrical stimulation induced synchronized ERK activation. Amplitude and duration of ERK activation were controlled by varying stimulation strength and duration. ERK activation frequencies were arbitrarily modulated with trains of short AC applications with accurately defined intervals. Significantly, ERK dynamics coded by well-designed AC can rewire PC12 cell fate independent of growth factors. This technique can be used to synchronize and modulate ERK activation dynamics, thus would offer a practical way to control cell behaviors  without the use of biochemical agents or genetic manipulation.

Electrically synchronizing and modulating the dynamics of ERK activation to regulate cell fate.

The development of wearable bioelectronic systems is a promising approach for optimal delivery of therapeutic treatments. These systems can provide continuous delivery of ions, charged biomolecules, and an electric field for various medical applications. However, rapid prototyping of wearable bioelectronic systems for controlled delivery of specific treatments with a scalable fabrication process is challenging. We present a wearable bioelectronic system comprised of a polydimethylsiloxane (PDMS) device cast in customizable 3D printed molds and a printed circuit board (PCB), which employs commercially available engineering components and tools throughout design and fabrication. The system, featuring solution-filled reservoirs, embedded electrodes, and hydrogel-filled capillary tubing, is assembled modularly. The PDMS and PCB both contain matching through-holes designed to hold metallic contact posts coated with silver epoxy, allowing for mechanical and electrical integration. This assembly scheme allows us to interchange subsystem components, such as various PCB designs and reservoir solutions. We present three PCB designs: a wired version and two battery-powered versions with and without onboard memory. The wired design uses an external voltage controller for device actuation. The battery-powered PCB design uses a microcontroller unit to enable pre-programmed applied voltages and deep sleep mode to prolong battery run time. Finally, the battery-powered PCB with onboard memory is developed to record delivered currents, which enables us to verify treatment dose delivered. To demonstrate the functionality of the platform, the devices are used to deliver H[Formula: see text] in vivo using mouse models and fluoxetine ex vivo using a simulated wound environment. Immunohistochemistry staining shows an improvement of 35.86% in the M1/M2 ratio of H[Formula: see text]-treated wounds compared with control wounds, indicating the potential of the platform to improve wound healing.

A system for bioelectronic delivery of treatment directed toward wound healing.

Wound healing is a complex physiological process that requires precise control and modulation of many parameters. Therapeutic ion and biomolecule delivery has the capability to regulate the wound healing process beneficially. However, achieving controlled delivery through a compact device with the ability to deliver multiple therapeutic species can be a challenge. Bioelectronic devices have emerged as a promising approach for therapeutic delivery. Here, we present a pro-reparative bioelectronic device designed to deliver ions and biomolecules for wound healing applications. The device incorporates ion pumps for the targeted delivery of H and zolmitriptan to the wound site. In vivo studies using a mouse model further validated the device's potential for modulating the wound environment via H delivery that decreased M1/M2 macrophage ratios. Overall, this bioelectronic ion pump demonstrates potential for accelerating wound healing via targeted and controlled delivery of therapeutic agents to wounds. Continued optimization and development of this device could not only lead to significant advancements in tissue repair and wound healing strategies but also reveal new physiological information about the dynamic wound environment.

A pro-reparative bioelectronic device for controlled delivery of ions and biomolecules.

Microglia play a critical role in neurodegenerative disorders, such as Alzheimer's disease, where alterations in microglial function may result in pathogenic amyloid-β (Aβ) accumulation, chronic neuroinflammation, and deleterious effects on neuronal function. However, studying these complex factors in vivo, where numerous confounding processes exist, is challenging, and until recently, in vitro models have not allowed sustained culture of critical cell types in the same culture.

Primary cortical cell tri-culture to study effects of amyloid-β on microglia function and neuroinflammatory response.

Electric fields (EFs) are widely employed to promote tissue regeneration and accelerate wound healing. Despite extensive study, the cellular responses elicited by EFs are complex and not well understood. The present work focuses on cell migration-a process essential to organismal development, immune surveillance, and repair-and seeks to achieve its precise, closed-loop regulation. Effective control is impeded by (i) the nonlinear and stochastic nature of migratory dynamics and (ii) safety constraints that restrict the admissible EF magnitude. To address these challenges, we reformulate a neural network (NN) feedback controller previously developed for single-cell membrane-potential regulation and adapt it to guide population-level cell migration. A projection operator is embedded into the NN weight-update law to prevent maladaptive learning that arises when the control signal saturates at its EF limit. Numerical simulations confirm that the modified controller maintains accurate trajectory tracking under saturation and outperforms the original NN design. Finally, we demonstrate a proof-of-concept by implementing the controller in vitro to direct the electrotactic migration of naïve macrophages in 2D culture under a unidirectional EF. For the in vitro experiments, we compare performance to the standard proportional-integral-derivative (PID) controller.

Controlling Cell Migratory Patterns Under an Electric Field Regulated by a Neural Network-Based Feedback Controller.

Wound healing is a dynamic process involving various cell types. Collecting samples from healing wounds and investigating their transcriptomics can provide deeper insights into the underlying processes. In recent years, several experiments have been conducted to gather transcriptomic data from wounds in both humans and animals. However, the temporal resolution of such data often does not adequately match the dynamics of the process, and spatial aspects are frequently overlooked. Here, we present a dataset collected from an experiment on wound healing in pigs, including gene expression profiles at the wound edge and center, and photographs of the wounds. Photographs provide non-invasive data, and advancements in image analysis using artificial intelligence methods are actively being integrated into medical practice. Being collected within the same experiment, these comprehensive data can aid in building intelligent wound diagnostics and treatment algorithms.

A high-resolution temporal transcriptomic and imaging dataset of porcine wound healing.

Wireless bioelectronic actuators have been developed to deliver targeted treatments over multiple days while continuously monitoring delivery, thereby improving wound healing. Specifically, these devices can deliver charged biomolecules such as fluoxetine cations (Flx[Formula: see text]) and electric field (EF) in freely moving pigs. Treatments can be controlled and monitored in real time via WiFi, with options for both user-specified delivery rates and durations, as well as automated closed-loop (CL) control. The devices are engineered to handle various failure scenarios that may arise in dynamic, real-world experiments-such as communication or power interruptions-ensuring that valuable experimental data is collected with minimal disruption. The ion pump features eight drug reservoirs and channels arranged around a 20 mm diameter-wound, with a central ground electrode (0 V). When voltages above 0 V are applied to the outer channels, currents flow from the reservoirs and channels into the wound, delivering Flx[Formula: see text] and/or EF depending on the reservoir solution. The device records applied voltages and currents locally to a microSD card at a high sampling rate, while simultaneously transmitting real-time measurements via a local WiFi network to a wound healing algorithm running on a nearby laptop. CL control of current/delivery rate is performed by an onboard microcontroller unit (MCU) and current-source microchips, based on instructions received from the wound healing algorithm. A graphical user interface (GUI) provides intuitive user control and real-time data visualization, with support for multiple devices. In vivo studies over seven days showed that Flx[Formula: see text]-treated wounds had a trend toward lower M1/M2 macrophage ratio and greater re-epithelialization compared to controls (standard-of-care), demonstrating the actuator's potential to enhance wound healing.

Article	Year
KCNJ15/Kir4.2 couples with polyamines to sense weak extracellular electric fields in galvanotaxis. Nature communications\| PubMed ID: 26449415	2015
cAMP and cGMP Play an Essential Role in Galvanotaxis of Cell Fragments. Journal of cellular physiology\| PubMed ID: 26517849	2016
An Experimental Model for Simultaneous Study of Migration of Cell Fragments, Single Cells, and Cell Sheets. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 27271908	2016
Electric fields accelerate cell polarization and bypass myosin action in motility initiation. Journal of cellular physiology\| PubMed ID: 28749047	2018
HLA-DRB1 polymorphisms and alopecia areata disease risk: A systematic review and meta-analysis. Medicine\| PubMed ID: 30095639	2018
Expression of integrins to control migration direction of electrotaxis. FASEB journal : official publication of the Federation of American Societies for Experimental Biology\| PubMed ID: 31116572	2019
Optimization of Electrical Stimulation for Safe and Effective Guidance of Human Cells. Bioelectricity\| PubMed ID: 34476366	2020
Electrically synchronizing and modulating the dynamics of ERK activation to regulate cell fate. iScience\| PubMed ID: 34746704	2021
A system for bioelectronic delivery of treatment directed toward wound healing. Scientific reports\| PubMed ID: 37679425	2023
A pro-reparative bioelectronic device for controlled delivery of ions and biomolecules. Wound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society\| PubMed ID: 38794912	2024
Primary cortical cell tri-culture to study effects of amyloid-β on microglia function and neuroinflammatory response. Journal of Alzheimer's disease : JAD\| PubMed ID: 39501607	2024
Controlling Cell Migratory Patterns Under an Electric Field Regulated by a Neural Network-Based Feedback Controller. Bioengineering (Basel, Switzerland)\| PubMed ID: 40722370	2025
A high-resolution temporal transcriptomic and imaging dataset of porcine wound healing. Scientific data\| PubMed ID: 41073450	2025
Development of a wireless bioelectronic actuator for wound healing in a porcine model. Scientific reports\| PubMed ID: 41203833	2025

Kan Zhu

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