Research Article

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

DOI:

10.3791/69666

⸱

December 19th, 2025

 ,  ,  ,  , 
1Department of Plastic Surgery, The First Affiliated Hospital of China Medical University, 2Department of Dermatology, Institute for Regenerative Cures, University of California, Davis, 3Department of Ophthalmology and Vision Science, Institute for Regenerative Cures, University of California, Davis

In This Article

Summary

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In a small cohort of female donors, aging reduced the electrotactic migration of hADSCs. DCEF altered gene expression (747 up, 624 down), enriching sodium transport/channel GO terms and PI3K-Akt KEGG pathways, suggesting involvement in age-related electrotaxis, without establishing causality.

Abstract

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Human adipose-derived stem cells (hADSCs) are pivotal for tissue regeneration and wound healing, and their directed migration is a prerequisite for exerting these therapeutic effects. Electric fields (EFs) are well-recognized as key cues guiding cell migration during wound repair, yet the electrotactic behavior of hADSCs -- especially how donor characteristics (e.g., age) regulate this behavior and its underlying molecular mechanisms -- remains poorly understood. This knowledge gap limits the optimized application of hADSCs in regenerative medicine. In this study, we first validated the existence of electrotaxis in hADSCs and confirmed its voltage dependence: under direct current electric fields (DCEFs) of 100-200 mV/mm, hADSCs migrated directionally toward the anode, with stronger EF intensities enhancing both migration directionality and speed (versus at 0 mV/mm control). We then compared hADSCs from young female donors (27.00 ± 4.58 years) and elderly female donors (62.33 ± 4.04 years) using RNA sequencing (RNA-seq) after 200 mV/mm DCEF stimulation. Transcriptomic analysis identified 747 upregulated and 624 downregulated genes in elderly hADSCs, with differentially expressed genes (DEGs) enriched in biological processes/pathways critical for electrotaxis -- including sodium ion transmembrane transport, voltage-gated sodium channel activity (GO terms), and the PI3K-Akt signaling pathway (KEGG pathway). Functionally, elderly hADSCs exhibited significantly reduced anodal migration (decreased accumulated distance, Euclidean distance, and directness) compared to young hADSCs under DCEF stimulation. This study provides the first evidence of age-dependent electrotaxis in hADSCs, demonstrating that donor age correlates with impaired electrotactic capacity. It further reveals that dysregulated sodium channel activity and PI3K-Akt signaling may underlie this age-related decline. These findings point to the potential regulatory mechanisms of hADSC electrotaxis and offer new insights for tailoring hADSC-based therapies (e.g., selecting optimal donors or targeting sodium/PI3K-Akt pathways) to improve tissue regeneration and wound healing outcomes.

Introduction

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Adipose-derived stem cells (ADSC) possess strong self-renewal capacity and significant regenerative potential. Under appropriate induction conditions, they can differentiate into osteoblasts, chondrocytes, and adipocytes, among others1. Through paracrine signaling, ADSC can also support the generation of blood vessels and lymphatic vessels2,3, inhibit scar formation4, and exhibit immune regulatory effects5. Therefore, ADSC is considered an ideal seed cell for cell-based therapies in regenerative medicine.

The function of ADSC is influenced by various donor characteristics, which may affect their potential efficacy in cell therapies, including donor age, body mass index, gender, and others6. ADSC derived from older donors exhibit more pronounced aging characteristics, with reduced adipogenic differentiation potential, osteogenic differentiation potential, and migratory capacity7,8,9. Furthermore, the ability to promote keratinocyte proliferation and stimulate the body's self-repair mechanisms is diminished10.

Cell-directed migration is a key physiological mechanism in wound healing and tissue regeneration, with electric fields (EFs) playing an important role in guiding cell migration and promoting wound repair11. For example, when the epithelial barrier is damaged, endogenous electric fields are immediately generated and persist until the wound heals and the epithelial barrier function is restored12,13,14. As one of the signals guiding directed cell migration, electric signals have been shown to have a higher priority than other coexisting signals, such as contact inhibition, mechanical forces, and chemotactic factors15,16. Several methods have been developed to promote wound healing and tissue regeneration, such as through drugs and external electric stimulation devices that enhance or mimic the endogenous electric fields in the human body17,18. The ability of cells to migrate according to an electric field gradient is known as electrotaxis. In vitro studies have shown that many different types of cells are capable of directed migration in electric fields. Some cells migrate toward the anode, such as mammalian cranial neural crest cells, human astrocytes, and human breast cancer cells19,20,21. Other cells migrate toward the cathode, such as human renal tubular epithelial cells, human dermal fibroblasts, and human neutrophils22,23,24. ADSC can be guided to migrate toward the anode in direct current (DC) electric fields, and under prolonged culture conditions, exogenous pulsed electric current has been shown to effectively promote the directional migration of ADSC toward the anode while maintaining cell viability25. To date, studies on the electrotaxis characteristics of human adipose-derived stem cells remain scarce, and the underlying mechanisms regulating their electrotactic behavior are still not fully understood.

We are the first to systematically investigate age-dependent electrotactic differences in hADSCs from young versus elderly female donors under gradient direct current electric fields (DCEF), and to further uncover the underlying mechanisms (involving sodium channel activity and PI3K-Akt signaling) using transcriptomic analysis. In this study, we examined the effects of DCEF on hADSCs. Using RNA sequencing and differential gene enrichment analysis, we compared gene expression profiles between two hADSC groups-derived from young and elderly female donors-following DCEF stimulation to identify age-related transcriptional differences. We hypothesized that donor aging impairs the electrotactic responsiveness of hADSCs, and that this age-related decline is linked to altered sodium channel activity and PI3K-Akt signaling.

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Protocol

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Informed consent was obtained from all participants prior to tissue collection. This study was approved by the Ethics Committee of the First Affiliated Hospital of China Medical University (Approval No.: [2018]2018-110-2). Written informed consent was obtained from all participants prior to tissue collection

hADSC isolation and culture
Tissue collection: Human abdominal adipose tissue samples were obtained during routine elective liposuction procedures performed at the Department of Plastic Surgery of the First Affiliated Hospital of China Medical University. All donors were healthy female volunteers undergoing cosmetic abdominal liposuction, and no additional surgical interventions were performed for research purposes. Tissue specimens were collected by the operating surgeon under sterile conditions immediately after aspiration and transferred to the laboratory in sterile containers on ice within 30 min.

Sample size: A total of 6 donors were included in this study, consisting of 3 younger donors (age: 27.00 ± 4.58 years) and 3 older donors (age: 62.33 ± 4.04 years).

Tissue processing: Transfer adipose tissue fragments into a 50 mL conical tube, centrifuge at 1,800 x g for 3 min at 4 °C, and carefully aspirate the upper oil phase (to remove excess lipids). Add 0.2% collagenase Type IV (final volume: 5-10 mL per 1 g adipose tissue) to the tissue pellet, then place the tube in a 37 °C water bath or incubator for 45 min. Gently agitate the tube every 10-15 min during digestion to ensure uniform contact between collagenase and tissue. After digestion, terminate the reaction by adding low-glucose DMEM supplemented with 10% FBS and 1% penicillin/streptomycin (volume ratio of neutralization medium to collagenase solution = 1:1).

CAUTION: When handling collagenase, wear disposable gloves, a lab coat, and safety goggles to avoid skin and eye contact, prepare and use the reagent in a biological safety cabinet (BSC) to prevent aerosol contamination, and in case of spillage, wipe up immediately with 75% ethanol and dispose of contaminated materials as biological waste.

Cell isolation: Centrifuge the neutralized digestion mixture at 1,200 x g for 10 min at 4 °C to pellet the cells. Discard the supernatant, then resuspend the pellet in erythrocyte lysis buffer (155 mM NH₄Cl, 10 mM KHCO₃, 0.1 mM EDTA; pH 7.4; volume: 2-3 mL per pellet) and incubate at room temperature (22-25 °C) for 5 min to lyse residual red blood cells. Filter the cell suspension sequentially through 200 µm strainers to remove undigested tissue debris and obtain a single-cell suspension.

Cell counting: Before seeding, cell numbers were quantified using a hemocytometer. Briefly, hADSCs were detached with 0.25% trypsin-EDTA, resuspended in growth medium, and mixed 1:1 with 0.4% trypan blue solution. Viable cells (non-stained) were counted manually using a Neubauer hemocytometer under a light microscope, and total viable cell numbers were calculated as: cell number/mL = (average counted cells x dilution factor x 104).

Cell culture: Seed the filtered cells at a density of 5 x 103 cells/cm2 in growth medium (low-glucose DMEM + 10% FBS + 1% penicillin/streptomycin) and incubate at 37 °C with 5% CO2. Replace the medium every 2-3 days to remove non-adherent cells and maintain optimal culture conditions. All hADSCs used for subsequent experiments -- including cell characterization (surface marker detection, trilineage differentiation assays), DCEF stimulation, migration and morphology analysis, senescence/proliferation assays, and RNA sequencing -- were at passage 3 to passage 5.

hADSC characterization
Expression of cell surface markers: Culture cells to 70% confluency, then dissociate adherent cells with 0.25% trypsin-EDTA (incubate at 37 °C for 3 min). Centrifuge the cell suspension at 300 x g for 5 min at 4 °C, discard the supernatant, and resuspend the cell pellet in 1x PBS (volume: 1-2 mL). Aliquot 1 x 105 cells per sample (resuspended in 100 µL of 1x PBS) and add fluorescently conjugated antibodies at the specified dilutions: anti-CD44 (1:50), anti-CD90 (1:100), anti-CD29 (1:50), anti-CD73 (1:50), and anti-CD105 (1:20)1,4,7. Incubate the antibody-cell mixture at 4 °C for 30 min in the dark to avoid fluorophore quenching. After incubation, centrifuge at 1,000 x g for 5 min at 4 °C and aspirate the supernatant completely. Resuspend the pellet in 1-2 mL of 1x PBS, centrifuge again at 1,000 x g for 3 min at 4 °C, discard the supernatant, and repeat this washing process 2x to remove unbound antibodies. Finally, resuspend the cell pellet in 200 µL of fresh 1x PBS, acquire fluorescence data using a flow cytometer, and perform quantitative analysis with FlowJo software (v10). The gating workflow followed standard MSC phenotyping procedures: FSC-SSC gate to exclude debris and select the main cell population based on size and granularity, followed by doublet exclusion using FSC-A vs. FSC-H and SSC-A vs. SSC-H plots to ensure single-cell events. Then, live cell gating was done to exclude trypan blue-positive or PI-positive dead cells. Fluorescence gating was done for single-stained controls, and unstained controls were used to set thresholds for CD29, CD44, CD73, CD90, CD105 (positive markers), and CD34, CD45 (negative markers). Representative gating plots were exported and analyzed to confirm the identity of hADSCs.

Differentiation potential
Adipogenic differentiation: Harvest P3-generation hADSCs at 85% confluency using 0.25% trypsin-EDTA, then seed them in 24-well plates at a density of 1 x 10⁵ cells per well (using complete DMEM medium) and incubate at 37 °C with 5% CO2 until cells reach 85% confluency again. For induction, use the adipogenesis differentiation kit, reconstituting kit component A (Adipogenic inducer) and component B (Adipogenic maintainer) as per the manual, and use component A for the first 3 days before switching to component B for 1 day. Repeat this cycle for 3 weeks. For staining, aspirate the induction medium, gently wash cells with 3 mL of 1x PBS (avoid disrupting cell layers), fix cells with 4% paraformaldehyde (PFA) at room temperature for 20 min, then incubate with 0.3% Oil Red O solution (prepared in 60% isopropanol) at room temperature for 15 min. Wash cells 3x with 1x PBS to remove excess stain, then image lipid droplets using an inverted light microscope (20x).

Osteogenic differentiation: Seed hADSCs in 12-well plates at a density of 1 x 10⁵ cells per well, and when cells reach 85% confluency, use the osteogenesis differentiation kit for induction -- reconstitute kit component A (Osteogenic inducer) and component B (Osteogenic supplement) in low-glucose DMEM (final concentrations: 10% FBS, 1% penicillin/streptomycin, 0.1 µM dexamethasone, 10 mM β-glycerophosphate, 0.05 mM ascorbic acid-2-phosphate) and refresh the medium every 3 days for 3 weeks. For staining, fix cells with 4% PFA at room temperature for 15 min, wash 3x with 1x PBS, then incubate with 2% Alizarin Red S solution (pH 4.2, prepared in deionized water) at room temperature for 30 min. Wash cells 3x with 1x PBS to remove unbound stain, then image calcium-deposited nodules under a phase-contrast microscope (20x) and quantify mineralization by measuring the area of Alizarin Red S-positive nodules using ImageJ software.

Chondrogenic differentiation: Centrifuge 250,000 hADSCs at 500 x g for 5 min at 4 °C in a 15 mL conical tube to form a cell pellet, add 500 µL of complete DMEM medium to the tube, and incubate overnight at 37 °C with 5% CO2 to stabilize the pellet. The next day, use the chondrogenesis differentiation kit for induction -- reconstitute kit component A (Chondrogenic inducer) in CO2-independent medium (final concentrations: 1% penicillin/streptomycin, 10 ng/mL TGF-β3, 50 µg/mL ascorbic acid-2-phosphate, 100 µg/mL sodium pyruvate) and gently refresh the medium every 3 days (avoid disturbing the pellet). After 3 weeks of induction, fix the cell pellet with 4% PFA at room temperature for 30 min, aspirate the fixative, incubate with 0.1% (w/v) Toluidine Blue O solution (prepared in 0.1 M sodium acetate buffer, pH 4.0) at room temperature for 15 min, aspirate the stain, rinse the pellet 3x with deionized water to remove excess dye, then image under a bright-field microscope (20x) and quantify sulfated proteoglycan accumulation by measuring the mean optical density (OD) of Toluidine Blue O staining using ImageJ software.

CAUTION: When handling 4% PFA (a toxic and corrosive reagent), work exclusively in a fume hood while wearing disposable gloves, a lab coat, and safety goggles to avoid inhalation of vapor or contact with skin and eyes -- if contact occurs, rinse immediately with running water for 15 min and seek medical attention. For hazardous waste disposal, collect used PFA and PFA-contaminated materials (e.g., pipette tips, plates) in a dedicated chemical waste container labeled PFA Waste and dispose of using institutional hazardous waste protocols, ensuring not to mix with biological waste.

Electrotaxis chamber assembly: Drill two holes (0.75 cm in diameter) at opposite ends of the centerline on a 10 cm Petri dish lid, positioned 1 cm from the dish edge (to accommodate agar-salt bridges). Apply a thin layer of vacuum grease (≤ 2 cm in length, ≤ 1 cm in width) on the bottom of the Petri dish along the centerline, 4 cm from each edge (Figure 1A). Using sterile fine-tipped forceps, place a sterile 1 cm x 2 cm glass coverslip on each grease patch, and press gently to ensure a tight seal (avoid coverslip breakage; Figure 1B-C). Apply another thin layer of vacuum grease on top of each attached coverslip, then place a second sterile 1 cm x 2 cm glass coverslip directly on the grease to form a double-coverslip stack. Press gently to ensure a tight seal between the two coverslips (prevent medium leakage; Figure 1D). Along the diagonal line connecting the top-left corner of one coverslip stack to the nearest dish edge, and the bottom-right corner of the opposite stack to its nearest edge, apply vacuum grease to form a continuous barrier wall. The wall should be tightly adhered to the dish bottom (no gaps) and measure approximately 2 cm in height and 0.5 cm in width (Figure 1E). Sterilize the assembled chamber under UV light for 20 min (to eliminate microbial contamination), then store at room temperature under sterile conditions until use (Figure 1F).

DCEF stimulation: Place sterile glass slides (for cell seeding) in a 10 cm Petri dish and incubate overnight at 37 °C with 5% CO2 to pre-condition the slides (promote cell adhesion). Prepare Steinberg's solution by diluting 10 mL of 10x stock solution in 90 mL of ddH2O (sterile; final concentrations: 58 mM NaCl, 0.67 mM KCl, 0.44 mM Ca(NO3)24H2O, 0.83 mM MgSO47H2O, 10 mM HEPES; pH 7.4). Add 0.3 g agar to 20 mL of the prepared Steinberg's solution, microwave for 20 s until the agar is fully dissolved and the solution is clear, then immediately fill custom glass salt bridges with the hot agar-Steinberg's mixture and allow it to solidify at room temperature. After solidification, sterilize the salt bridges under UV light for 15 min, and sterilize two beakers (each containing 30 mL of Steinberg's solution) under UV light for 15 min. Seed P3-generation hADSCs on the pre-conditioned sterile slides at a density of 2 x 10⁴ cells/cm², incubate at 37 °C with 5% CO2 for 24 h to allow cell adhesion, then transfer the cell-seeded slides to the runway of the assembled electrotaxis chamber and secure them with sterile side slides (to prevent movement). Seal the runway by placing a 3 cm x 2 cm glass coverslip over the vacuum grease, pressing gently to ensure a tight seal, and applying additional vacuum grease along the edges of the top coverslip to form a barrier (prevent medium evaporation). Fill the chamber's reservoirs with CO2-independent medium (supplemented with 10% FBS and 1% penicillin/streptomycin; volume: 5-8 mL per reservoir), insert the sterilized agar-salt bridges through the holes in the Petri dish lid (with one end immersed in the chamber's medium reservoir and the other end immersed in the Steinberg's solution-filled beakers), and connect the beakers to a power supply through Ag/AgCl electrodes (to deliver a stable direct current). Apply a DC electric field (DCEF) of 2 V/cm for 4 h, monitor the voltage every 15 min using a digital multimeter (adjust if necessary to maintain constant field strength), and capture time-lapse images of cell migration every 5 min at 4 random fields using the software (5x objective, bright-field mode).

NOTE: When using electrical equipment, ensure the power supply and electrodes are dry and placed on a non-conductive surface to avoid electric shock. Wear insulated gloves when connecting or disconnecting electrodes, turn off the power before adjusting the setup, and in case of medium leakage onto electrical components, turn off the power immediately and wipe up with absorbent paper soaked in 75% ethanol. In this study, by minimizing the thickness of the electrophoresis chamber and maintaining it at approximately 1 mm, along with conducting multi-point voltage measurements at both ends of the chamber, the variation in field strength was controlled within 10%. Under these conditions, it is considered that the electric field distribution within the culture area is essentially uniform26.

Migration and morphology analysis
Live imaging: For hADSCs under DCEF stimulation, import the time-lapse image sequences (5-min intervals) into ImageJ software, manually track 100 cells across 4 independent fields from the start (t=0) to the end of stimulation (t=4 h) using the Manual Tracking plugin, and calculate the mean migration velocity (µm/min) and directionality (D/T ratio, where D = straight-line distance from start to end, T = total path length) using the Chemotaxis Tool plugin (ImageJ).

Tracking: Calculate the following migration parameters to quantify electrotactic behavior: Directedness (Σcosθi/n, where θi is the angle between the cell's displacement vector and the direction of the electric field (EF), and n is the total number of tracked cells, ranging from -1 to 1 with values closer to 1 indicating stronger anode-directed migration), Accumulated Distance (total path length traveled by each cell during the stimulation period, in µm), Track Speed (accumulated distance divided by stimulation time, in µm/h), and Euclidean Distance (straight-line start-to-end displacement of each cell, in µm, reflecting net migration).

Morphometry: After DCEF stimulation, fix cells with 4% PFA at room temperature for 20 min, wash 3x with 1x PBS, stain the cytoskeleton with Phalloidin-TRITC (1:500 dilution in 1x PBS; volume: 200 µL per well for 24-well plates) at room temperature for 30 min (to label F-actin), and counterstain nuclei with DAPI (1 µg/mL in 1x PBS; volume: 100 µL per well) for 5 min. Cells were imaged using a fluorescence microscope at 20x magnification (with representative high-resolution images captured at 40x). Phalloidin-TRITC was visualized using an excitation wavelength of 540-555 nm and an emission wavelength of 565-580 nm, while DAPI was imaged using an excitation wavelength of 358-405 nm and an emission wavelength of 420-480 nm. Then measure the following morphological parameters in ImageJ: Long Axis (maximum Feret diameter, the longest distance between any two points on the cell perimeter), Vertical Length (width of the cell perpendicular to the long axis), and Verticality (sinα, where α is the angle between the cell's long axis and the direction of the EF, with higher values indicating greater alignment with the EF).

Senescence and proliferation assays
SA-β-Gal staining: Use a senescence detection kit to identify senescent cells (blue-stained cells) under a bright-field microscope (20x objective). Seed P3-generation hADSCs in 6-well plates at a density of 2 x 105 cells per well, culture until 80% confluency, aspirate the medium, gently wash cells 3x with 1x PBS, add 1 mL of Fixative Solution (provided in the kit) per well and incubate at room temperature for 15 min, wash cells 3x with 1x PBS to remove residual fixative after fixation, add 0.5 mL of SA-β-gal Staining Working Solution (prepared by mixing 50 µL of SA-β-gal substrate with 450 µL of staining buffer per the kit manual) per well, seal the plate with transparent film (to prevent evaporation), incubate overnight (16-18 h) at 37 °C (avoid CO2 exposure, as it alters pH and inhibits enzyme activity), capture bright-field images of ≥10 random fields per well (10x objective) the next day, count the number of blue-stained (SA-β-gal+) cells and total cells, and calculate the percentage of senescent cells as (Number of SA-β-gal+ cells / Total number of cells) x 100.

Ki67 immunostaining
After SA-β-gal staining, aspirate the staining solution, wash cells 2x with 1x PBS, permeabilize cell membranes with 0.1% Triton X-100 (prepared in 1x PBS; 1 mL per well) at room temperature for 10 min, and block non-specific antibody binding with 5% BSA (in 1x PBS; 1 mL per well) at room temperature for 1 h. Cells were then incubated with Anti-Ki67 primary antibody (1:200 dilution in 1% BSA/PBS; 500 µL per well) at 4 °C overnight. The next day, cells were washed 3x with 1x PBS (5 min each) remove unbound primary antibody and incubated with Alexa Fluor 488 488-conjugated secondary antibody (1:500 dilution in 1% BSA/PBS; 500 µL per well) at room temperature for 1 h in the dark. Cells were washed again 3x with 1x PBS and counterstained with DAPI (1 µg/mL in 1x PBS; 200 µL per well) for 5 min. Fluorescence images were captured using a fluorescence microscope at 20x magnification (with representative high-resolution images acquired at 40x). Alexa Fluor 488-labeled Ki67+ cells were visualized using an excitation wavelength of 485-495 nm and an emission wavelength of 515-545 nm, while DAPI-stained nuclei were imaged using an excitation wavelength of 358-405 nm and an emission wavelength of 420-480 nm. The percentage of proliferative cells was calculated as:(Number of Ki67+ cells / Number of DAPI+ nuclei) x 100.

NOTE: When handling primary and secondary antibodies, work in a BSC to prevent contamination, aliquot antibodies into single-use volumes to avoid repeated freeze-thaw cycles, and collect antibody-contaminated materials (e.g., pipette tips, plates) in a dedicated autoclavable Biological Waste bag for sterilization by autoclaving before disposal.

Age-dependent electrotaxis analysis of hADSCs: Use a DC pulse power supply to generate DCEF of three intensities: 0 mV/mm (control), 100 mV/mm, and 200 mV/mm, employ a live-cell workstation to observe and record cell migration in real time, and for each EF intensity, quantitatively compare the anodal migration capacity of hADSCs from young and elderly female donors by measuring Accumulated Distance, Euclidean Distance, Track Speed, and Directedness, with three independent biological replicates per group to ensure result reliability. Electrotactic migration parameters were quantified using the Manual Tracking and Chemotaxis Tool plugins in ImageJ (NIH). Individual cells were manually tracked for the entire 4-h stimulation period, and the following parameters were computed according to standard methods: Accumulated Distance: the total path length traveled by each cell during the recording period. Euclidean Distance: the straight-line distance between the cell's starting and ending positions. Track Speed: accumulated distance divided by total tracking time (µm/h). Directedness: calculated as the mean cosine of the angle (θ) between each displacement vector and the electric-field vector (values range from -1 to 1; values closer to 1 indicate strong anodal migration).

RNA sequencing
RNA extraction and preparation: Sterilize an ice-filled container under UV light for 15 min (to maintain RNA stability) and perform all subsequent steps on ice. Transfer hADSC-seeded glass slides (1 cm x 2 cm) from the electrotaxis chamber to a sterile Petri dish, wash cells 3x with 3 mL of ice-cold 1x PBS (add PBS gently, agitate slightly, and aspirate completely to remove residual medium), then add 1 mL of TRIzol reagent to each slide and incubate at room temperature for 20 min to fully lyse cells (ensure the lysate covers the entire cell layer). Transfer the lysate to an RNase-free 1.5-mL centrifuge tube, add 0.2 mL of chloroform, vortex vigorously for 15 s to induce phase separation, incubate the tube on ice for 15 min, then centrifuge at 12,000 x g for 15 min at 4 °C. This separates the lysate into three layers: upper aqueous phase containing RNA, middle protein layer, and lower organic phase. Carefully transfer the upper aqueous phase (≈ 0.5 mL) to a new RNase-free tube (avoid touching the middle layer), add 0.5 mL of isopropanol, vortex gently to precipitate RNA, incubate on ice for 10 min, then centrifuge at 12,000 x g for 10 min at 4 °C; RNA will form a white pellet at the bottom of the tube. Discard the supernatant, repeat the isopropanol precipitation once to improve RNA purity, aspirate the supernatant completely, air-dry the RNA pellet in a BSC at room temperature for 5-10 min (do not over-dry, as this reduces solubility), resuspend the RNA pellet in 30 µL of DEPC-treated water, incubate at 55 °C for 10 min to aid dissolution, quantify RNA purity by measuring the A260/A280 ratio (target: 1.8-2.0) using a spectrophotometer, assess RNA integrity using a RNA Nano Chip; target: RNA Integrity Number [RIN] > 8.0, and store qualified RNA samples at -80 °C until sequencing.

CAUTION: TRIzol and chloroform are toxic, volatile, and carcinogenic, so handle them exclusively in a fume hood while wearing nitrile gloves, a lab coat, and safety goggles -- avoid inhalation of vapors, wipe with a paper towel and rinse with soap and water for 10 min if spilled on skin, and do not mix it with bleach or other oxidizing agents (as this may produce toxic gases). For hazardous waste disposal related to RNA extraction, collect mixtures, isopropanol supernatants, and contaminated tubes in a sealed, chemically resistant container labeled RNA Waste (do not mix with aqueous waste or biological waste) and dispose of following institutional hazardous waste protocols, ensuring not to pour down drains.

Data preprocessing and quality control: Prepare sequencing libraries using the VAHTS mRNA-seq Library Prep Kit following the manufacturer's protocol -- enrich mRNA using oligo(dT) magnetic beads, fragment mRNA into 200-300 bp fragments using divalent cations, synthesize first-strand cDNA using random hexamers followed by second-strand cDNA, perform end repair, add A-tails, ligate sequencing adapters, amplify libraries by PCR, and purify using beads. Sequence the libraries on a sequencing platform (150 bp paired end reads), generating approximately 50 million raw reads per sample, then use a software to trim low-quality reads (remove reads with >50% bases having Phred quality score <20) and adapter sequences, retaining approximately 48 million clean reads per sample (Q30 > 90%) for downstream analysis after trimming.

Bioinformatics analysis: Align the clean reads to the human reference genome (GRCh38/hg38) using Hisat2 v2.2.1 software and save alignment results as BAM files, use htseq-count v0.13.5 software to count the number of reads mapped to each gene (based on Ensembl gene annotations), normalize gene count data using the estimateSizeFactors function from the DESeq (2012) R package to eliminate batch effects and differences in sequencing depth, and perform differential expression analysis using the nbinomTest function (DESeq package), selecting differentially expressed genes (DEGs) using thresholds of fold change (FC) > 2 and adjusted p-value (padj) < 0.05. Conduct Gene Ontology (GO) enrichment analysis (biological process, molecular function, cellular component) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis on DEGs using clusterProfiler v4.0 R package, focusing on enrichments related to cell migration (e.g., cell adhesion, cell migration), ion transport (e.g., sodium ion transmembrane transport), and signal pathways (e.g., PI3K-Akt signaling pathway). Perform unsupervised hierarchical clustering on DEGs using pheatmap v1.0.12 R package and visualize gene expression patterns across samples with a heatmap (row-scaled z-scores).

Statistical analysis: All experimental data are presented as Mean ± Standard Error of the Mean (Mean ± SEM), with at least 3 independent biological replicates per group (n ≥ 3). Use Student's t-test to compare differences between two groups (e.g., young vs. elderly donors) and one-way Analysis of Variance (ANOVA) followed by Tukey's post-hoc test to compare differences among three or more groups (e.g., different EF intensities), performing statistical analyses using SPSS 23.0 software and defining statistical significance as follows: *p < 0.05, **p < 0.01, ***p < 0.001.

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Results

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Application of electric fields guides the directional migration of hADSCs and enhances their migratory capacity
The extracted hADSCs exhibited typical mesenchymal stem cell surface markers (e.g., CD29, CD44, CD90, CD73, CD105) and possessed trilineage differentiation potential (adipogenic, osteogenic, chondrogenic; Figure 2A), meeting the standard criteria for hADSC identification. We applied direct current electric fields (DCEFs) at intensities of 0 mV/mm, 100 mV/mm, an...

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Discussion

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Early in the tissue injury repair and regeneration process, electric fields emerge as one of the earliest physical cues established in the microenvironment. Their directional properties are spatiotemporally correlated with the recruitment of cells to the injury site27. Endogenous electric fields originate from the transepithelial potential generated by polarized ion transport in epithelial tissues. When the epithelial barrier is disrupted or undergoes aging, the potential is secondarily altered, w...

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Disclosures

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The authors have no conflicts of interest to disclose.

Acknowledgements

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We sincerely acknowledge the Department of Plastic Surgery at The First Affiliated Hospital of China Medical University (Shenyang, China) and the Institute for Regenerative Cures at the University of California, Davis (CA, USA) for their provision of critical experimental infrastructure and shared research resources that enabled this study. We also extend our gratitude to Shanghai OE Biotech Co., Ltd. for their technical support in RNA sequencing and bioinformatic analysis.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
4°C/-20°C/-80°C RefrigeratorHaier, ChinaHYC-390/DW-25L262/DW-86L728J
AgarHUSHI, China10000561
Agilent 2100 BioanalyzerAgilent Technologies, United StatesG2939BA
Alizarin Red S Sigma-Aldrich, United StatesA5533
Biosafety Cabinet Thermo Fisher Scientific, United States1300 A2 1.5 m
CaNO3·4H2OHUSHI, China10013960
CentrifugeThermo Fisher Scientific, United StatesX4R Pro 
CO2 IncubatorThermo Fisher Scientific, United StatesForma Steri-Cycle 3307 
Collagenase Type IVBiosharp, China BS076
Confocal MicroscopeZEISS, GermanyLSM 900
DC Pulse Power Supply Daedo Powertronics, KoreaDDP-500
DexamethasoneSigma-Aldrich, United StatesD4902
Digital MultimeterFluke, United StatesFLUKE-175
DMSOSigma-Aldrich, United StatesD2650
Electronic Balance Sartorius, Germany BSA224S-CW
FBS Gibco, United States10270-106
Flow CytometerThermo Fisher Scientific, United StatesA24858
IBMX (1-Methyl-3-isobutylxanthine)Sigma-Aldrich, United StatesI7018
Indomethacin  Sigma-Aldrich, United StatesI7378
InsulinSigma-Aldrich, United StatesI5500  
Inverted MicroscopeZEISS, GermanyAxio Observer 7 / ACR
ITS-GSigma-Aldrich, United StatesI3146
KClHUSHI, China10019318
KH2PO4HUSHI, China10019320
Linoleic Acid Sigma-Aldrich, United StatesL1376
Live Cell StationLive Cell Instrument, KoreaChamlide TC 
Low-glucose DMEMGibco, United States11885084
MgSO4·7H2O HUSHI, China10019322
Na2HPO4·12H2OHUSHI, China10019324
NaClHUSHI, China10019318
NanoDrop 2000 SpectrophotometerThermo Fisher Scientific, United StatesND-2000
Oil Red OSigma-Aldrich, United StatesO0625
Penicillin/StreptomycinGibco, United States15140122
PipettesEppendorf, Germany 4920000061
SYBR Premix EX TaqTakara, China RR420A
Toluidine Blue O     Sigma-Aldrich, United StatesT3260
TrisHUSHI, China10019326
TRIzolBeyotime, ChinaR0016
Trypsin-EDTAGibco, United States25200056
Upright MicroscopeZEISS, Germany415200-0000-000 
Vacuum GreaseDOW CORNING, United States1597418
Vitamin C   Sigma-Aldrich, United StatesA4544
Water Purification SystemThermo Fisher Scientific, United States GenPure xCAD Plus UF-TOC
β-Glycerophosphate Sigma-Aldrich, United StatesG9422

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Adipose Derived Stem CellsElectrotaxisElectric Field StimulationAge Dependent MigrationStem Cell MigrationRNA SequencingSodium Channel ActivityPI3K Akt SignalingTissue RegenerationWound Healing

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